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

Summary

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.

Abstract

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.

Introduction

The hyperemic response to a brief period of tissue ischemia has emerged as a key non-invasive measure of (micro)vascular function. During occlusion of a conduit artery, downstream arterioles dilate in an effort to offset the ischemic insult. Upon release of the occlusion, the decreased vascular resistance results in hyperemia, the magnitude of which is dictated by one's ability to dilate the downstream microvasculature. While reactive hyperemia is a strong independent predictor of cardiovascular events^1,2 and therefore a clinically significant endpoint, its functional significance to exercise tolerance and quality of life is less clear.

Indeed, dynamic exercise represents a major cardiovascular stress that demands a highly coordinated neurovascular response in order to match oxygen delivery to metabolic demand. For example, skeletal muscle blood flow can increase nearly 100-fold during isolated muscle contractions³, which would overwhelm the pumping capacity of the heart if such a hemodynamic response were extrapolated to whole-body exercise. Accordingly, to avoid severe hypotension, sympathetic (i.e., vasoconstrictor) nervous activity increases to redistribute cardiac output away from inactive and visceral tissues and towards active skeletal muscle⁴. Sympathetic outflow is also directed to the exercising skeletal muscle⁵; however, local metabolic signaling attenuates the vasoconstrictor response in order to ensure adequate tissue oxygen delivery^{6,7,8,9,10,11}. Collectively, this process is termed functional sympatholysis¹², and is imperative to the normal regulation of skeletal muscle blood flow during exercise. Since skeletal muscle blood flow is a key determinant of aerobic capacity — an independent predictor of quality of life and cardiovascular disease morbidity and mortality¹³— understanding the control of skeletal muscle blood flow and tissue oxygen delivery during exercise is of great clinical significance.

Oxygen delivery is only half of the Fick equation, however, with oxygen utilization satisfying the other half of the equation. Among the major determinates of oxygen utilization, mitochondrial oxidative phosphorylation plays an essential role in supplying adequate energy for cellular processes both at rest and during exercise. Indeed, impairments in muscle oxidative capacity can limit functional capacity and quality of life^14,15,16. Various measures are commonly used to provide an index of muscle oxidative capacity, including invasive muscle biopsies and expensive and time-consuming magnetic resonance spectroscopy (MRS) techniques.

Here, we propose a novel, non-invasive approach, using near-infrared spectroscopy (NIRS), to assess each of these three major clinical endpoints (reactive hyperemia, sympatholysis, and muscle oxidative capacity) in a single clinic or laboratory visit. The major advantages of this approach are three-fold: First, this technique is easily portable, relatively low cost, and easy to perform. Current Doppler ultrasound approaches for measuring reactive hyperemia are highly operator-dependent — requiring extensive skill and training — and require sophisticated, high-cost, data acquisition hardware and post-processing software. Moreover, this could conceivably be introduced into the clinic and/or large clinical trials for bedside monitoring or testing therapeutic efficacy. Second, by virtue of the methodology, this technique focuses specifically on the skeletal muscle microvasculature, increasing the overall specificity of the technique. Alternative approaches using Doppler ultrasound focus entirely on upstream conduit vessels and infer changes downstream, which can dampen the signal. Third, this technique is completely non-invasive. Skeletal muscle oxidative capacity is traditionally assessed with invasive and painful muscle biopsies, and functional sympatholysis may be assessed with intra-arterial injection of sympathomimetics and sympatholytics. This approach avoids these requirements all together.

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Protocol

This protocol follows the guidelines of the institutional review board at the University of Texas at Arlington and conforms to the standards set by the latest version of the Declaration of Helsinki. Accordingly, written informed consent was (and should be) obtained prior to commencement of research procedures.

1. Instrumentation

NOTE: The following instrumentation description is based on the near-infrared (NIR) spectrometer and data acquisition system used in our lab (see Table of Materials). Thus, the instructions include steps that are necessary for the optimal function of these devices. These steps include the calibration of the NIR probe using the accompanying software and calibration phantom, and the application of a dark cloth to exclude ambient light. In the event that different data collection hardware and/or software are used, investigators should consult their own specific user manuals for calibration and ambient light considerations. Figure 1 illustrates the experimental set-up and instrumentation described immediately below.

1. Instruct the subject to lie supine with their legs inside a lower body negative pressure (LBNP) chamber (Figure 1A), so that their belt line is approximately even with the opening to the LBNP box. For instructions on how to build a LBNP chamber, see References¹⁷.
2. Place three electrocardiogram electrodes on the subject: two in an inferior, mid-clavicular location and one on the subject's left side medial to the iliac crest. This configuration provides the best results due to limited access to the lower limbs, instrumentation of the upper limbs, and arm movement during hand grip exercise.
3. Place a non-invasive blood pressure monitor module on the subject's dominant wrist. Place the finger blood pressure cuffs on each finger and connect them to the module (Figure 1B). Ensure the finger blood pressure cuffs are properly calibrated according to the user's manual accompanying your device.
4. Instruct the subject to grasp a hand grip dynamometer (HGD) with their non-dominant arm in a slightly abducted position. The arm should be comfortably positioned on a bedside table. The distance and angle of the HGD should be adjusted to allow for optimal grip strength with minimal arm movement (Figure 1C).
5. Secure the HGD to a bedside table.
6. Measure the maximum voluntary contraction (MVC) of the participant. Tell the participant that, when prompted, they must squeeze the HGD as hard as possible while only utilizing the muscles in the hand and forearm. Instruct the subject that they must refrain from recruiting their upper arm, chest, shoulder, or abdominal muscles when performing the maximum grip.
7. Repeat Step 1.6 three times, separated by at least 60 s. Record the maximum force achieved (best of 3). This maximum force will be used to calculate the exercise intensity for skeletal muscle oxidative capacity and neurovascular coupling (below).
8. Place a rapid-inflation cuff around the upper arm of the exercising hand. Connect the airline from the rapid inflation controller to the cuff.
9. Identify the flexor digitorum profundus. Use a skin marker to demarcate the borders of the palpable muscle.
10. Ensure that the NIR spectrometer is properly calibrated according to the user's manual included with your device. Clean the skin over which the NIR probe will be positioned with an alcohol prep wipe.
11. Place the NIR probe over the center of the belly of the muscle (flexor digitorum profundus) and affix it securely to the forearm.
12. Wrap the probe and forearm with dark cloth, minimizing interference from ambient light (Figure 1C, Figure 1D).
13. When ready to perform the functional sympatholysis portion of the study, seal the subject into the LBNP chamber.

2. Skeletal Muscle Oxidative Capacity

NOTE: A representative data tracing illustrating the experimental procedure for measuring skeletal muscle oxidative capacity is depicted in Figure 2. This experimental approach has previously been validated against in vivo phosphorus MRS¹⁸ and in situ muscle respirometry¹⁹, and is gaining widespread acceptance²⁰.

1. Instrument the subject as indicated above (Instrumentation).
2. Instruct the subject to lie still for 2 min while monitoring deoxyhemoglobin (HHb) and oxyhemoglobin (HbO₂) via the NIR probe.
   NOTE: This rest period allows the subject to recover from any movement artifact associated with the instrumentation process, and ensures stable baseline measurements. If after 2 min no significant fluctuations have occurred, the subject may be considered at a steady state, or resting baseline.
3. Prior to cuff occlusion, notify your subject that you will be inflating the cuff. Inflate the upper arm cuff at least 30 mmHg above systolic blood pressure for 5 min (i.e., suprasystolic). Instruct the subject to keep their arm as still and relaxed as possible both during cuff inflation and following cuff deflation.
   NOTE: This 5 min brachial artery cuff occlusion protocol closely reflects the currently accepted clinical standard for vascular occlusion tests^{21,22,23,24,25}.
4. Record the initial/baseline value (prior to cuff occlusion) and the nadir value of tissue saturation (StO₂) during the cuff occlusion and determine the midpoint between these two values.
   
5. Allow the subject to recover from the cuff occlusion and return to the resting baseline values. Once the subject has maintained a resting baseline for at least 1 full min, continue to the next step.
6. Instruct subject to squeeze and maintain an isometric hand grip at 50% of their MVC. Encourage the subject to maintain their isometric contraction until the tissue desaturates by 50%. Upon achieving this value, tell the subject to relax their hand and inform them that no more exercise or movement is needed.
7. Within 3 - 5 s following exercise cessation, administer the following rapid cuff occlusion series (one series = 1 inflation + 1 deflation), as previously established¹⁸:
   Series #1 - 6: 5 s on/5 s off
   Series #7 - 10: 7 s on/10 s off
   Series #11 - 14: 10 s on/15 s off
   Series #15 - 18: 10 s on/20 s off
8. After completing the 18^th inflation/deflation series, instruct the subject to rest, allowing tissue saturation to return to initial baseline values. After these values have remained consistent for at least 2 min, repeat steps 2.4 and 2.5.
9. Calculating Skeletal Muscle Oxidative Capacity
   1. Calculate the slope of change in the StO₂for each of the individual 18 cuff occlusions, forming the monoexponential recovery points illustrated in Figure 2C.
   2. Fit the calculated data from 2.7 to the following monoexponential curve^18,19,26
      y = End - Δ x e^-kt
      NOTE: 'y' is the relative muscle oxygen consumption rate (mV̇O₂) during cuff inflation, 'End' represents the mV̇O₂ immediately following the cessation of exercise; delta ('Δ') signifies the change in mV̇O₂ from rest to the end of exercise; 'k' is the fitting rate constant; 't' is time. Tau is calculated as 1/k.

3. Reactive Hyperemia

NOTE: A representative data tracing illustrating the experimental procedure for measuring reactive hyperemia is depicted in Figure 3.

1. With the subject lying supine and instrumented as described above (Instrumentation), instruct the subject to lie as still as possible.
2. Once the subject has achieved a consistent resting state, continue to record at least 1 min of baseline data and then rapidly inflate a blood pressure cuff on the upper arm to a suprasystolic pressure (30 mmHg above systolic blood pressure).
3. At the 5 min mark, rapidly deflate the cuff while recording the hyperemic response.
4. Continue recording for at least 3 min to capture the subject's recovery.
5. Calculating Reactive Hyperemia
   NOTE: The NIRS parameters calculated are depicted in Figure 3.
   1. Calculate baseline StO₂ as the average StO₂ over 1 full min prior to the onset of arterial cuff occlusion.
   2. Determine the resting skeletal muscle metabolic rate as the desaturation rate (i.e., average slope) during cuff occlusion (defined as Slope 1)^27,28.
   3. Calculate reactive hyperemia as follows:
      a) the average upslope following cuff release (i.e., reperfusion rate, defined as slope 2), calculated from the moment of cuff release through the linearly increasing phase of the rebound trace;
      b) the highest StO₂ value reached after cuff release (denoted as StO_2max);
      c) the reactive hyperemia area under the curve (AUC); calculated from the time of cuff release to 1-, 2- and 3-min post cuff-occlusion (AUC 1-min, AUC 2-min, and AUC 3-min, respectively); and
      d) the hyperemic reserve, calculated as the change in StO₂ above baseline and reported as a percent (%) change. This value is calculated as the highest saturation achieved during the post-occlusive rebound minus the average saturation calculated in step 3.5.1 (see above).
      NOTE: Large differences in baseline data will greatly affect the interpretation of the hyperemic reserve.

4. Functional Sympatholysis

NOTE: A representative data tracing illustrating the experimental procedure for measuring functional sympatholysis is depicted in Figure 4.

1. Instrument the subject as indicated above (Instrumentation).
2. Ensure an airtight seal in the LBNP chamber.
3. With the subject lying still and at rest, collect 3 min of baseline data.
4. At the 3 min mark, turn on the vacuum. Adjust the vacuum so that the pressure inside the LBNP chamber is between -20 and -30 mmHg. Allow the vacuum to run for 2 min while monitoring the subject's response.
5. At the 5 min mark, turn off the vacuum and allow the subject to rest for 3 min.
6. At the 8 min mark, initiate the voice prompt guiding the subject through the rhythmic hand grip exercise (20% MVC).
7. Confirm that the subject is maintaining their squeeze throughout the entirety of each gripping phase and relaxing completely during between each repetition. Monitor their force output and confirm that they are achieving 20% MVC with each grip. Continue exercise until the 11 min mark.
8. At the 11 min mark, turn on the vacuum encouraging the subject to continue their rhythmic exercise. Allow the vacuum to run from 11 - 13 min, then turn it off.
9. Have the subject continue performing rhythmic hand grip exercise at 20% of their MVC for an additional 2 min. Upon exercise cessation, have the subject rest quietly and lie still.
10. Calculating Functional Sympatholysis
    1. Normalize the change in oxyhemoglobin with LBNP to the total labile signal (TLS), determined during 5 min cuff occlusion:
       
       
    2. Calculate each event as the final 20 min average of each event.
    3. Calculate the exercise-induced attenuation of the oxyhemoglobin reduction:
       

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Results

Skeletal muscle oxidative capacity

Figure 2 illustrates a representative participant response during a NIRS-derived skeletal muscle oxidative capacity assessment. Panel A shows the tissue saturation profile during a 5 min arterial cuff occlusion protocol, handgrip exercise, and intermittent arterial occlusion during recovery from exercise. Panel B illustrates the expected t...

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Discussion

The methods described herein enable non-invasive, clinical evaluation of reactive hyperemia, neurovascular coupling, and skeletal muscle oxidative capacity in a single clinic or laboratory visit.

Critical Considerations

Although NIRS is relatively robust and easy to use, collection of these data require careful placement of the optodes directly over the muscle belly, secured tightly in place to avoid movement artifact, and covered with a bl...

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Materials

Name	Company	Catalog Number	Comments
Dual-channel OxiplexTS Near-infrared spectroscopy machine	Iss Medical	101
NIRS muscle sensor	Iss Medical	201.2
E20 Rapid cuff inflation system	Hokanson	E20
AG101 Air Source	Hokanson	AG101
Smedley Handgrip dynometer (recording)	Stolting	56380
Powerlab 16/35, 16 Channel Recorder	ADInstruments	PL3516
Human NIBP Set	ADInstruments	ML282-SM
Bio Amp	ADInstruments	FE132
Quad Bridge Amp	ADInstruments	FE224
Connex Spot Monitor	Welch Allyn	71WX-B
Origin(Pro) graphing software	OrignPro	Pro
Lower body negative pressure chamber	Physiology Research Instruments	standard unit