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="NIRS protocol diagram; supine rest, baseline data, thigh cuff inflation, occlusion, hyperemia phases." class="xfigimg" src="/files/ftp_upload/66511/66511fig01.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
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 °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's recommendations, researchers' 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'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'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="Blood flow restriction training setup, arm compression cuffs for exercise physiology research." class="xfigimg" src="/files/ftp_upload/66511/66511fig02.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
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="Electrode placement for neuromuscular stimulation study on forearm; experiment setup with electrodes." class="xfigimg" src="/files/ftp_upload/66511/66511fig03v3.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
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 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'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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<li>Tucker, W. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Studies+into+the+determinants+of+skeletal+muscle+oxygen+consumption%3A+Novel+insight+from+near-infrared+diffuse+correlation+spectroscopy%5BTitle%5D)+AND+%22J+Physiol-London%22%5BJournal%5D)">Studies into the determinants of skeletal muscle oxygen consumption: Novel insight from near-infrared diffuse correlation spectroscopy.</a> J Physiol-London. 597 (11), 2887-2901 (2019).</li>
<li>Tucker, W. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Near-infrared+diffuse+correlation+spectroscopy+tracks+changes+in+oxygen+delivery+and+utilization+during+exercise+with+and+without+isolated+arterial+compression%5BTitle%5D)+AND+%22Am+J+Physiol+Regul+Integr+Comp+Physiol%22%5BJournal%5D)">Near-infrared diffuse correlation spectroscopy tracks changes in oxygen delivery and utilization during exercise with and without isolated arterial compression.</a> Am J Physiol Regul Integr Comp Physiol. 318 (1), R81-R88 (2020).</li>
<li>Wassenaar, E. B., Van Den Brand, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reliability+of+near-infrared+spectroscopy+in+people+with+dark+skin+pigmentation%5BTitle%5D)+AND+%22J+Clinic+Monit+Comput%22%5BJournal%5D)">Reliability of near-infrared spectroscopy in people with dark skin pigmentation.</a> J Clinic Monit Comput. 19 (3), 195-199 (2005).</li>
<li>Soares, R. N., Murias, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Near-infrared+spectroscopy+assessment+of+microvasculature+detects+difference+in+lower+limb+vascular+responsiveness+in+obese+compared+to+lean+individuals%5BTitle%5D)+AND+%22Microvasc+Res%22%5BJournal%5D)">Near-infrared spectroscopy assessment of microvasculature detects difference in lower limb vascular responsiveness in obese compared to lean individuals.</a> Microvasc Res. 118, 31-35 (2018).</li>
<li>Boezeman, R. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Monitoring+of+foot+oxygenation+with+near-infrared+spectroscopy+in+patients+with+critical+limb+ischemia+undergoing+percutaneous+transluminal+angioplasty%3A+A+pilot+study%5BTitle%5D)+AND+%22Eur+J+Vasc+Endovasc+Surg%22%5BJournal%5D)">Monitoring of foot oxygenation with near-infrared spectroscopy in patients with critical limb ischemia undergoing percutaneous transluminal angioplasty: A pilot study.</a> Eur J Vasc Endovasc Surg. 52 (5), 650-656 (2016).</li>
<li>Lin, B. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Using+wireless+near-infrared+spectroscopy+to+predict+wound+prognosis+in+diabetic+foot+ulcers%5BTitle%5D)+AND+%22Adv+Skin+Wound+Care%22%5BJournal%5D)">Using wireless near-infrared spectroscopy to predict wound prognosis in diabetic foot ulcers.</a> Adv Skin Wound Care. 33 (1), 1-12 (2020).</li>
<li>Weingarten, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diffuse+near-infrared+spectroscopy+prediction+of+healing+in+diabetic+foot+ulcers%3A+A+human+study+and+cost+analysis%5BTitle%5D)+AND+%22Wound+Repair%22%5BJournal%5D)">Diffuse near-infrared spectroscopy prediction of healing in diabetic foot ulcers: A human study and cost analysis.</a> Wound Repair. 20 (2), A44-A44 (2012).</li>
<li>Murrow, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Near-infrared+spectroscopy-guided+exercise+training+for+claudication+in+peripheral+arterial+disease%5BTitle%5D)+AND+%22Eur+J+Prev+Cardiol%22%5BJournal%5D)">Near-infrared spectroscopy-guided exercise training for claudication in peripheral arterial disease.</a> Eur J Prev Cardiol. 26 (5), 471-480 (2018).</li>
</ol>

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><tbody><tr><td>Cuff Inflator Air Source</td><td>Hokanson&amp;nbsp;</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&amp;nbsp;</td><td>E20 RAPID CUFF INFLATOR</td><td></td></tr><tr><td>Thigh Cuff</td><td>Hokanson&amp;nbsp;</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.

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 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, the ...

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

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

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.

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. ...

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

Research

JoVE Journal

Biology

863 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.

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

Dual-mode Imaging of Cutaneous Tissue Oxygenation and Vascular Function

Accurate assessment of cutaneous tissue oxygenation and vascular function is important for appropriate detection, staging, and treatment of many health disorders such as chronic wounds. We report the development of a dual-mode imaging system for non-invasive and non-contact imaging of cutaneous tissue oxygenation and vascular function. The imaging system integrated an infrared camera, a CCD camera, a liquid crystal tunable filter and a high intensity fiber light source. A Labview interface was programmed for equipment control, synchronization, image acquisition, processing, and visualization. Multispectral images captured by the CCD camera were used to reconstruct the tissue oxygenation map. Dynamic thermographic images captured by the infrared camera were used to reconstruct the vascular function map. Cutaneous tissue oxygenation and vascular function images were co-registered through fiduciary markers. The performance characteristics of the dual-mode image system were tested in humans.

A dual-mode imaging system was developed for non-contact assessment of cutaneous tissue oxygenation and vascular function.

Accurate assessment of cutaneous tissue oxygenation and vascular function is important for appropriate detection, staging, and treatment of many health ...

Medicine

Non-invasive Assessment of Microvascular and Endothelial Function

The authors have utilized capillaroscopy and forearm blood flow techniques to investigate the role of microvascular dysfunction in pathogenesis of cardiovascular disease. Capillaroscopy is a non-invasive, relatively inexpensive methodology for directly visualizing the microcirculation. Percent capillary recruitment is assessed by dividing the increase in capillary density induced by postocclusive reactive hyperemia (postocclusive reactive hyperemia capillary density minus baseline capillary density), by the maximal capillary density (observed during passive venous occlusion). Percent perfused capillaries represents the proportion of all capillaries present that are perfused (functionally active), and is calculated by dividing postocclusive reactive hyperemia capillary density by the maximal capillary density. Both percent capillary recruitment and percent perfused capillaries reflect the number of functional capillaries. The forearm blood flow (FBF) technique provides accepted non-invasive measures of endothelial function: The ratio FBFmax/FBFbase is computed as an estimate of vasodilation, by dividing the mean of the four FBFmax values by the mean of the four FBFbase values. Forearm vascular resistance at maximal vasodilation (FVRmax) is calculated as the mean arterial pressure (MAP) divided by FBFmax. Both the capillaroscopy and forearm techniques are readily acceptable to patients and can be learned quickly.
The microvascular and endothelial function measures obtained using the methodologies described in this paper may have future utility in clinical patient cardiovascular risk-reduction strategies. As we have published reports demonstrating that microvascular and endothelial dysfunction are found in initial stages of hypertension including prehypertension, microvascular and endothelial function measures may eventually aid in early identification, risk-stratification and prevention of end-stage vascular pathology, with its potentially fatal consequences.

Capillaroscopy is a non-invasive, relatively inexpensive methodology for directly visualizing the microcirculation. The forearm blood flow technique provides accepted non-invasive measures of endothelial function.

The authors have utilized capillaroscopy and forearm blood flow techniques to investigate the role of microvascular dysfunction in pathogenesis of ...

Exploring Cognitive Functions in Babies, Children &amp; Adults with Near Infrared Spectroscopy

An explosion of functional Near Infrared Spectroscopy (fNIRS) studies investigating cortical activation in relation to higher cognitive processes, such as language1,2,3,4,5,6,7,8,9,10, memory11, and attention12 is underway worldwide involving adults, children and infants 3,4,13,14,15,16,17,18,19 with typical and atypical cognition20,21,22. The contemporary challenge of using fNIRS for cognitive neuroscience is to achieve systematic analyses of data such that they are universally interpretable23,24,25,26, and thus may advance important scientific questions about the functional organization and neural systems underlying human higher cognition.
Existing neuroimaging technologies have either less robust temporal or spatial resolution. Event Related Potentials and Magneto Encephalography (ERP and MEG) have excellent temporal resolution, whereas Positron Emission Tomography and functional Magnetic Resonance Imaging (PET and fMRI) have better spatial resolution. Using non-ionizing wavelengths of light in the near-infrared range (700-1000 nm), where oxy-hemoglobin is preferentially absorbed by 680 nm and deoxy-hemoglobin is preferentially absorbed by 830 nm (e.g., indeed, the very wavelengths hardwired into the fNIRS Hitachi ETG-400 system illustrated here), fNIRS is well suited for studies of higher cognition because it has both good temporal resolution (~5s) without the use of radiation and good spatial resolution (~4 cm depth), and does not require participants to be in an enclosed structure27,28. Participants cortical activity can be assessed while comfortably seated in an ordinary chair (adults, children) or even seated in mom s lap (infants). Notably, NIRS is uniquely portable (the size of a desktop computer), virtually silent, and can tolerate a participants subtle movement. This is particularly outstanding for the neural study of human language, which necessarily has as one of its key components the movement of the mouth in speech production or the hands in sign language. 
The way in which the hemodynamic response is localized is by an array of laser emitters and detectors. Emitters emit a known intensity of non-ionizing light while detectors detect the amount reflected back from the cortical surface. The closer together the optodes, the greater the spatial resolution, whereas the further apart the optodes, the greater depth of penetration. For the fNIRS Hitachi ETG-4000 system optimal penetration / resolution the optode array is set to 2cm. 
Our goal is to demonstrate our method of acquiring and analyzing fNIRS data to help standardize the field and enable different fNIRS labs worldwide to have a common background.

Exploring Cognitive Functions in Babies, Children & Adults with Near Infrared Spectroscopy

Here we describe a data collection and data analysis method for functional Near Infrared Spectroscopy (fNIRS), a novel non-invasive brain imaging system used in cognitive neuroscience, particularly in studying child brain development. This method provides a universal standard of data acquisition and analysis vital to data interpretation and scientific discovery.

An explosion of functional Near Infrared Spectroscopy (fNIRS) studies investigating cortical activation in relation to higher cognitive processes, such as ...

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching morphogenesis. We have developed the limb skin vasculature model to study vascular development in which a pre-existing primitive capillary plexus is reorganized into a hierarchically branched vascular network. Whole-mount confocal microscopy with multiple labelling allows for robust imaging of intact blood vessels as well as their cellular components including endothelial cells, pericytes and smooth muscle cells, using specific fluorescent markers. Advances in this limb skin vasculature model with genetic studies have improved understanding molecular mechanisms of vascular development and patterning. The limb skin vasculature model has been used to study how peripheral nerves provide a spatial template for the differentiation and patterning of arteries. This video article describes a simple and robust protocol to stain intact blood vessels with vascular specific antibodies and fluorescent secondary antibodies, which is applicable for vascularized embryonic organs where we are able to follow the process of vascular development.

We introduce a whole-mount immunohistochemistry and laser scanning confocal microscopy with multiple labelling for analyzing intricate vascular network formation in mouse embryonic limb skin.

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching ...

Skeletal Muscle Neurovascular Coupling, Oxidative Capacity, and Microvascular Function with 'One Stop Shop' Near-infrared Spectroscopy

Exercise represents a major hemodynamic stress that demands a highly coordinated neurovascular response in order to match oxygen delivery to metabolic demand. Reactive hyperemia (in response to a brief period of tissue ischemia) is an independent predictor of cardiovascular events and provides important insight into vascular health and vasodilatory capacity. Skeletal muscle oxidative capacity is equally important in health and disease, as it determines the energy supply for myocellular processes. Here, we describe a simple, non-invasive approach using near-infrared spectroscopy to assess each of these major clinical endpoints (reactive hyperemia, neurovascular coupling, and muscle oxidative capacity) during a single clinic or laboratory visit. Unlike Doppler ultrasound, magnetic resonance images/spectroscopy, or invasive catheter-based flow measurements or muscle biopsies, our approach is less operator-dependent, low-cost, and completely non-invasive. Representative data from our lab taken together with summary data from previously published literature illustrate the utility of each of these end-points. Once this technique is mastered, application to clinical populations will provide important mechanistic insight into exercise intolerance and cardiovascular dysfunction.

Here, we describe a simple, non-invasive approach using near-infrared spectroscopy to assess reactive hyperemia, neurovascular coupling and skeletal muscle oxidative capacity in a single clinic or laboratory visit.

Exercise represents a major hemodynamic stress that demands a highly coordinated neurovascular response in order to match oxygen delivery to metabolic ...

Collection, Isolation and Enrichment of Naturally Occurring Magnetotactic Bacteria from the Environment

Magnetotactic bacteria (MTB) are aquatic microorganisms that were first notably described in 19751 from sediment samples collected in salt marshes of Massachusetts (USA). Since then MTB have been discovered in stratified water- and sediment-columns from all over the world2. One feature common to all MTB is that they contain magnetosomes, which are intracellular, membrane-bound magnetic nanocrystals of magnetite (Fe3O4) and/or greigite (Fe3S4) or both3, 4. In the Northern hemisphere, MTB are typically attracted to the south end of a bar magnet, while in the Southern hemisphere they are usually attracted to the north end of a magnet3,5. This property can be exploited when trying to isolate MTB from environmental samples.
One of the most common ways to enrich MTB is to use a clear plastic container to collect sediment and water from a natural source, such as a freshwater pond. In the Northern hemisphere, the south end of a bar magnet is placed against the outside of the container just above the sediment at the sediment-water interface. After some time, the bacteria can be removed from the inside of the container near the magnet with a pipette and then enriched further by using a capillary racetrack6 and a magnet. Once enriched, the bacteria can be placed on a microscope slide using a hanging drop method and observed in a light microscope or deposited onto a copper grid and observed using transmission electron microscopy (TEM).
Using this method, isolated MTB may be studied microscopically to determine characteristics such as swimming behavior, type and number of flagella, cell morphology of the cells, shape of the magnetic crystals, number of magnetosomes, number of magnetosome chains in each cell, composition of the nanomineral crystals, and presence of intracellular vacuoles.

We demonstrate a method to collect magnetotactic bacteria (MTB) that can be applied to natural waters. MTB can be isolated and enriched from sediment samples using a relatively simple setup that takes advantage of the bacteria's natural magnetism. Isolated MTB can then be examined in detail using both light and electron microscopy.

Magnetotactic bacteria (MTB) are aquatic microorganisms that were first notably described in 19751 from sediment samples collected in salt marshes of ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Cerebrovascular Reactivity Measurement with Functional Near Infrared Spectroscopy

Cerebrovascular reactivity (CVR) is the capacity of blood vessels in the brain to alter cerebral blood flow (either with dilation or constriction) in response to chemical or physical stimuli. The amount of reactivity in the cerebral microvasculature depends on the integrity of the capacitance vasculature and is the primary function of endothelial cells. CVR is, therefore, an indicator of the microvasculature&#8217;s physiology and overall health. Imaging methods that can measure CVR are available but can be costly, and require magnetic resonance imaging centers and technical expertise. In this study, we used fNIRS technology to monitor changes of oxyhemoglobin (HbO) and deoxyhemoglobin (HbR) in the cerebral microvasculature to assess the CVR of 15 healthy controls (HC) in response to a vasoactive stimulus (inhaled 5% carbon dioxide or CO2). Our results suggest that this is a promising imaging technology that offers a non-invasive, accurate, portable, and cost-effective method of mapping cortical CVR and associated microvasculature function, resulting from a traumatic brain injury or other conditions associated with cerebral microvasculopathy.

Presented here is a protocol for imaging and measurement of cerebrovascular reactivity in humans with functional Near Infrared Spectroscopy (fNIRS). fNIRS is a novel imaging modality that captures the concentration changes of hemoglobin species in the brain&#8217;s outermost cortex under specific stimuli.

Cerebrovascular reactivity (CVR) is the capacity of blood vessels in the brain to alter cerebral blood flow (either with dilation or constriction) in ...

Neuroscience

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of central nervous system injuries in vivo, possibly by reducing oxidative stress. However, effects of R/NIR-LT on oxidative stress have been shown to vary depending on wavelength or intensity of irradiation. Studies comparing treatment parameters are lacking, due to absence of commercially available devices that deliver multiple wavelengths or intensities, suitable for high through-put in vitro optimization studies. This protocol describes a technique for delivery of light at a range of wavelengths and intensities to optimize therapeutic doses required for a given injury model. We hypothesized that a method of delivering light, in which wavelength and intensity parameters could easily be altered, could facilitate determination of an optimal dose of R/NIR-LT for reducing reactive oxygen species (ROS) in vitro.
Non-coherent Xenon light was filtered through narrow-band interference filters to deliver varying wavelengths (center wavelengths of 440, 550, 670 and 810nm) and fluences (8.5 x 10-3 to 3.8 x 10-1 J/cm2) of light to cultured cells. Light output from the apparatus was calibrated to emit therapeutically relevant, equal quantal doses of light at each wavelength. Reactive species were detected in glutamate stressed cells treated with the light, using DCFH-DA and H2O2 sensitive fluorescent dyes.&#160;
We successfully delivered light at a range of physiologically and therapeutically relevant wavelengths and intensities, to cultured cells exposed to glutamate as a model of CNS injury. While the fluences of R/NIR-LT used in the current study did not exert an effect on ROS generated by the cultured cells, the method of light delivery is applicable to other systems including isolated mitochondria or more physiologically relevant organotypic slice culture models, and could be used to assess effects on a range of outcome measures of oxidative metabolism.

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...

Non-Invasive Monitoring of Microvascular Oxygenation and Reactive Hyperemia using Hybrid, Near-Infrared Diffuse Optical Spectroscopy for Critical Care

The detection of levels of impairment in microvascular oxygen consumption and reactive hyperemia is vital in critical care. However, there are no practical means for a robust and quantitative evaluation. This paper describes a protocol to evaluate these impairments using a hybrid near-infrared diffuse optical device. The device contains modules for near-infrared time-resolved and diffuse correlation spectroscopies and pulse-oximetry. These modules allow the non-invasive, continuous, and real-time measurement of the absolute, microvascular blood/tissue oxygen saturation (StO2) and the blood flow index (BFI) along with the peripheral arterial oxygen saturation (SpO2). This device uses an integrated, computer-controlled tourniquet system to execute a standardized protocol with optical data acquisition from the brachioradialis muscle. The standardized vascular occlusion test (VOT) takes care of the variations in the occlusion duration and pressure reported in the literature, while the automation minimizes inter-operator differences. The protocol we describe focuses on a 3-min occlusion period but the details described in this paper can readily be adapted to other durations and cuff pressures, as well as other muscles. The inclusion of an extended baseline and post-occlusion recovery period measurement allows the quantification of the baseline values for all the parameters and the blood/tissue deoxygenation rate that corresponds to the metabolic rate of oxygen consumption. Once the cuff is released, we characterize the tissue reoxygenation rate, magnitude, and duration of the hyperemic response in BFI and StO2. These latter parameters correspond to the quantification of the reactive hyperemia, which provides information about the endothelial function. Furthermore, the above-mentioned measurements of the absolute concentration of oxygenated and deoxygenated hemoglobin, BFI, the derived metabolic rate of oxygen consumption, StO2, and SpO2 provide a yet-to-be-explored rich data set that can exhibit disease severity, personalized therapeutics, and management interventions.

We describe a protocol to non-invasively and continuously measure absolute microvascular blood flow index and blood oxygen saturation using a multi-modal device based on near-infrared diffuse optics. We then evaluate the metabolic rate of oxygen consumption and reactive hyperemia utilizing a vascular occlusion test.

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

Summary

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Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Skeletal Muscle Neurovascular Coupling, Oxidative Capacity, and Microvascular Function with 'One Stop Shop' Near-infrared Spectroscopy

Collection, Isolation and Enrichment of Naturally Occurring Magnetotactic Bacteria from the Environment

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Cerebrovascular Reactivity Measurement with Functional Near Infrared Spectroscopy

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