The specific goals of this experiment are to noninvasively assess skeletal muscle oxidative capacity, to assess exercise skeletal muscle blood flow regulation, and to measure reactive hyperemia. These methods can help answer important questions related to the pathophysiology of exercise intolerance and cardiovascular disease. The main advantage of this technique is that we can noninvasively assess macro and micro vascular function and skeletal oxidative capacity in any clinical or laboratory setting.

Generally, individuals new to this method will struggle because of the operator-dependent nature of near-infrared spectroscopy. It is important to achieve consistent and accurate placement of the probe, as small deviations from optimal placement could have a large impact on signal quality. Begin with instructing the subject to lie supine with their legs inside an LBNP chamber so that their belt line is approximately even with the opening of the LBNP box.

Next, optimally place three electrocardiogram electrodes on the subject, two in an inferior midclavicular location and one on the subject's left side medial to the iliac crest. To proceed, place a non-invasive blood pressure monitor module on the subject's dominant wrist. Make use of properly-calibrated finger blood pressure cuffs.

Place them on each finger. Now instruct the subject to grasp an HGD with their nondominant hand in a slightly abducted position. Have the subject position their arm comfortably on the bedside table, then adjust the distance and angle of the HGD for optimal grip strength with minimal arm movement.

Then secure the HGD to a bedside table. Next, measure the maximum voluntary contraction of the subject. Tell the participant that when prompted to squeeze the HGD as hard as possible, only utilizing the muscles in the hand and forearm.

The subject must refrain from recruiting their upper arm, chest, shoulder, or abdominal muscles. Take three measurements separated by 60 seconds and record the highest force exerted. Next, place a rapid inflation cuff around the upper arm of the exercising hand.

Connect the air line from the rapid inflation controller to the cuff. Then palpate for the flexor digitorum profundus and mark its borders. Then clean the skin over the muscle with an alcohol prep wipe and place the NIR probe over the center of the belly of the muscle.

Proper identification of the flexor digitorum profundus is crucial for the correct placement of the near-infrared probe. To do this, instruct the subject to perform a light grip on the dynamometer while palpating the anterior medial aspect of the forearm. Affix the probe securely to the forearm and wrap it with dark cloth to minimize interference from ambient light.

After completing the instrumentation, instruct the subject to lie still for two minutes and record the initial baseline value of tissue saturation prior to the cuff occlusion. Then inflate the upper arm cuff to at least 30 millimeters of mercury above systolic for five minutes. It is very important that the subject be still and relaxed during all cuff inflations and deflations to minimize motion artifacts.

Now record the lowest value of tissue saturation during the cuff occlusion. After deflating the cuff, let the subject return to the resting baseline values. Once the subject has maintained a resting baseline for at least one full minute, instruct the subject to squeeze and maintain an isometric hand grip at 50%of their maximum voluntary contraction.

Encourage the subject to maintain their isometric contraction until the tissue desaturates by 50%Then tell the subject to relax their hand and that no more exercise or movement is needed. Within three to five seconds, administer a rapid cuff occlusion series consisting of 18 inflations and deflations of progressively greater durations. After the 18th series, instruct the subject to rest and stay as still as possible.

Once saturation has returned to baseline for at least two minutes, repeat the 18 cuff occlusions once more. Instrument the subject as before and seal the subject into the LBNP chamber. Ensure that the seal is airtight.

Then with the subject lying still and at rest collect three minutes of baseline data. At the three minute mark, turn on the vacuum to drop the chamber pressure by 20 to 30 millimeters of mercury. Run the vacuum for two minutes while monitoring the subject's response.

At the five minute mark, turn off the vacuum and allow the subject to rest for three minutes. Then at the eight minute mark, initiate a voice prompt guiding the subject through a rhythmic hand grip exercise at 20%maximum voluntary contraction. Observe that the subject is maintaining a consistent squeeze at 20%of maximum and relaxing completely between grips.

Do this until the 11 minute mark. Then turn on the vacuum while the subject continues the exercise. After two minutes turn off the vacuum and have the subject continue the exercise for two more minutes.

When this is over, have the subject rest quietly and lie still. The NIRS-derived skeletal muscle oxidative capacity assessment includes a tissue saturation profile during a five minute arterial cuff occlusion protocol, hand grip exercise, and intermittent arterial occlusion during recovery from exercise. The expected tissue desaturation-resaturation profile shows that during the intermittent arterial occlusions during the recovery period, the rate of desaturation is directly proportional to the rate of muscle oxygen consumption.

The calculated muscle oxygen consumption recovery data should fit to a mono-exponential curve and the recovery time constant derived. The NIRS-derived reactive hyperemia profile provides valuable insight into vascular reactivity and the test is easily adaptable and clinically meaningful. In a healthy control subject, when LBNP is superimposed on a mild hand grip the reflex decrease in muscle oxygenation is attenuated by about 50%Failure to attenuate sympathic nerve activity during exercise disrupts the balance between oxygen delivery and utilization, and causes functional muscle ischemia.

After watching this video you should have a good understanding of how to noninvasively assess skeletal muscle oxidative capacity, reactive hyperemia, and functional sympatholysis using commercially available near-infrared spectroscopy. While attempting this procedure, it's important to allow subjects to recover adequately between each stimulus. Once mastered, this data collection can be completed in 60 minutes or less.

Following this procedure, other methods like diffuse correlation spectroscopy which assesses microvascular perfusion, can be incorporated to provide further mechanistic insight into skeletal muscle oxygen delivery and utilization.