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J Appl Physiol 89: 1868-1878, 2000;
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Vol. 89, Issue 5, 1868-1878, November 2000

Regional blood flow during exercise in humans measured by near-infrared spectroscopy and indocyanine green

Robert Boushel1, Henning Langberg1, Jens Olesen1, Markus Nowak1, Lene Simonsen2, Jens Bülow2, and Michael Kjær1

1 Sports Medicine Research Unit and 2 Department of Clinical Physiology, Copenhagen Muscle Research Centre, Bispebjerg Hospital, Copenhagen, Denmark


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Using near-infrared spectroscopy (NIRS) and the tracer indocyanine green (ICG), we quantified blood flow in calf muscle and around the Achilles tendon during plantar flexion (1-9 W). For comparison, blood flow in calf muscle was determined by dye dilution in combination with magnetic resonance imaging measures of muscle volume, and, for the peritendon region, blood flow was measured by 133Xe washout. From rest to a peak load of 9 W, NIRS-ICG blood flow in calf muscle increased from 2.4 ± 0.2 to 74 ± 5 ml · 100 ml tissue-1 · min-1, similar to that measured by reverse dye (77 ± 6 ml · 100 ml tissue-1 · min-1). Achilles peritendon blood flow measured by NIRS-ICG rose with exercise from 2.2 ± 0.5 to 15.1 ± 0.2 ml · 100 ml-1 · min-1, which was similar to that determined by 133Xe washout (2.0 ± 0.6 to 14.6 ± 0.3 ml · 100 ml tissue-1 · min-1). This is the first study using NIRS and ICG to quantify regional tissue blood flow during exercise in humans. Due to its high spatial and temporal resolution, the technique may be useful for determining regional blood flow distribution and regulation during exercise in humans.

microvascular perfusion; spectrophotometry muscle; tendon


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

A NEED EXISTS FOR VERSATILE methods to study regional blood flow distribution and its regulation in humans during exercise. Indicator-dilution methods, plethysmography, and, more recently, ultrasound Doppler have been used to determine blood flow in the limbs during exercise. However, these methods do not specify patterns of blood flow in discrete regions within a limb or muscle group. Radioisotope tracer methods have been applied in humans; however, systematic errors in the clearance method may introduce inaccuracies in muscle tissue (2, 13), and individuals may be exposed to unnecessary risk. Ethanol removal with microdialysis has been used for local blood flow estimates in muscle (14), but increases in blood flow may not be detected without the influence of muscle contraction (30). Recently, it has been possible to quantify regional muscle perfusion during exercise using magnetic resonance imaging (MRI) (11). Despite some limitations, including movement artifact, this method provides high spatial and temporal resolution and the potential to detect perfusion heterogeneity during exercise.

Near-infrared (NIR) light easily penetrates biological tissue and allows for detection of changes in specific chromophore concentrations in human tissue. Changes in the NIR spectroscopy (NIRS) signal attributed to hemoglobin saturation emerge primarily from the absorption of light in arterioles, capillaries, and venules (6). The differential light absorption between large and small blood vessels is described by Beer's law, whereby photons successfully migrate through tissue regions with minimal absorbance. Thus, in the microcirculation, light absorption is small, allowing for multiple complete passage of photons along their pathway and therefore detection of chromophore absorption changes.

By using NIRS and a light-absorbing tracer, it is possible to measure blood flow by applying the Fick principle. The rate of accumulation of a tracer in a given tissue is equal to its rate of inflow minus its rate of outflow. If a tracer is introduced rapidly and its rate of accumulation is measured over time, blood flow can be measured as a ratio of the tracer accumulated to the quantity of tracer introduced over a given time.

NIRS has been used previously to measure cerebral blood flow under resting conditions by using oxygen as a tracer, and good agreement has been found with the 133Xe washout technique (4, 9, 34). However, this method is impractical for measuring muscle blood flow during exercise. NIRS was first applied with indocyanine green (ICG) as a tracer to measure cerebral blood flow at rest in the duck, and a good correlation was also found with measurements made by 133Xe washout (7). Recently, this technique has been validated with other methods for resting cerebral blood flow measurements in human infants (21, 27) and in pigs (15). Although ICG curves have been recorded previously in muscle by use of NIRS (24), this methodology has yet to be applied to quantify muscle blood flow during exercise in either humans or animals.

ICG is a water-soluble tricarbocyanine, light-absorbing dye with a peak absorption in human blood in the NIR range (800 nm). It has been used routinely for measuring cardiac output as well as limb blood flow with use of photodensitometry (12). After intravascular injection, ICG is predominantly bound to albumin (19) and is metabolized rapidly by hepatic parenchymal cells making it ideal for repeated blood flow measurements.

The purpose of this study was to 1) determine if the NIRS-ICG technique could be applied to quantify blood flow during exercise, 2) examine its sensitivity for differentiating blood flow in different tissues, and 3) compare the methodology against other established blood flow methods. Blood flow was measured in calf muscle and Achilles peritendon regions at rest and during graded plantar flexion exercise. Unit volume blood flow of calf muscle measured by NIRS-ICG was compared with that determined by dye dilution in combination with MRI measures of leg volume, and the NIRS-ICG blood flow in the Achilles region was compared with that determined by 133Xe washout. We hypothesized that NIRS could be used as an accurate and sensitive tool to measure blood flow during steady-state leg exercise.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Five young, healthy individuals participated in the study after informed consent as approved by the Ethical Committee of Copenhagen (KF) 01-392/98. Under local anesthesia, a catheter was inserted in the right femoral artery according to the Seldinger technique, and another catheter was similarly placed retrogradely in the popliteal vein of the same leg guided by ultrasound B-mode imaging of the vessel. Another venous catheter was placed into a vein of the right arm for injection of ICG (see below).

Cardiac output and blood pressure. Cardiac output was measured by the ICG (Cardio-Green; Passel & Lorel, Hanau, Germany) dye-dilution method (12). Five milligrams of dye were injected rapidly into the arm vein from a calibrated glass syringe followed by a 5-ml flush of isotonic saline. Blood from the femoral artery was withdrawn with a pump (Harvard, 2202A) at 20 ml/min through a linear photodensitometer connected to a cardiac output computer (Waters CO-10, Rochester, MN) for measurement of the arterial dye concentration (Fig. 1). The dye curves were displayed on a chart recorder (Gould 8000) and extrapolated with a logarithmic scale based on the exponential decay (downslope) observed from 75 to 50% of the peak dye concentration to correct for recirculation. Withdrawn arterial blood was reinfused into the arm vein. Cardiac output was then computed as the ratio of dye injected to the average arterial ICG concentration ([ICG]) over the time interval of the curve and expressed per minute. After each experiment, an ICG calibration curve was derived by measuring the voltage deflection from three separate samples of blood from each subject with known concentrations of ICG added. The arterial ICG curves were also used to quantify the input function for calculation of the regional tissue blood flow with NIRS (see NIRS-ICG blood flow).


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  		Fig. 1.   Schematic of the experimental setup for plantar flexion exercise. NIRS, near-infrared spectroscopy.

Blood pressure was determined from a pressure transducer (T100209A, Baxter, Unterschleissheim, Germany) positioned at the level of the heart, connected to the arterial catheter and interfaced to an on-line monitor (Dialogue 2000, Danica, Copenhagen, Denmark) and chart recorder. Mean arterial pressure was determined as the integrated pressure wave curve over time. Tissue vascular conductances were determined as the ratio of NIRS-ICG blood flow in calf muscle and peritendon regions to the mean arterial pressure, correcting for the vertical displacement of the pressure transducers from the level of the lower leg.

Muscle blood flow by dye dilution and MRI. The standard method used to determine calf muscle blood flow in this study was the dye-dilution method in combination with MRI measurements of muscle volume. The dye-dilution procedure and calculation of limb blood flow were exactly the same as for cardiac output, except that ICG was injected in the femoral artery and blood was withdrawn from the popliteal vein. ICG (0.5-1.0 mg) was rapidly injected into the femoral artery, followed by a 5-ml flush of saline. Blood from the popliteal vein was withdrawn at 10-15 ml/min through the photodensitometer (as described above), and the dye-dilution curves were recorded similarly on the chart recorder. The dye calibration curves derived from whole blood were used to calculate blood flow as described above for cardiac output.

The dye-dilution method yields blood flow values across the whole lower leg. Therefore, to express blood flow per unit volume of the calf muscle for comparison with the NIRS-ICG method, MRI was applied to determine the volume of the various tissues in the lower leg of each subject. Leg blood flow was thereby expressed as milliliters per 100 milliliters per minute. MRI scans were performed with a Siemens 1.5-T Magnetom vision scanner (Siemens, Germany), with 28 continuous axial spin lattice relaxation time-weighted images of the right lower leg obtained in a multislice spin-echo FLASH sequence (repetition time = 500 ms, echo time = 15 ms) using a body coil. Slice thickness was 3 mm with 15-mm interslice gap. Pixel size was 1 mm2. This setting was selected to optimize image quality to clearly separate muscle, bone, fat, and connective tissue. The same sequence, but with half the field of view and half the slice gap, was used for visualization of the Achilles tendon.

During MRI, subjects were in a supine position with the lower legs resting on a pillow. Image analysis and definition of anatomical cross sections were performed by use of NIH Image software (25). After manual outlining of each anatomical compartment of interest in each slice, the anatomical cross-sectional area was measured by computerized pixel counting within each compartment in each slice. The volumes of the compartments were calculated as the sum of each anatomical cross-sectional area times the distance covered by each slice (1.5 and 0.9 cm for muscle and tendon, respectively).

Achilles peritendon blood flow. The standard method used for blood flow measurements in the Achilles peritendon region was the 133Xe-washout technique. 133Xe was dissolved in sterile isotonic saline at a concentration of ~10 MBq/ml, and 0.1 ml of this solution was injected at a depth of 2 cm directly into the tissue ventral to the Achilles tendon, 5 cm proximal to the upper medial portion of the calcaneal insertion of the leg. A portable scintillation detector was secured to the peritendinous region over the 133Xe depot directly over the NIR probes (Fig. 1). The detector was connected to a multichannel analyzer system (Oakfield Instruments, Oxford, UK), and blood flow was calculated as
Blood flow<IT>=</IT>−100·&lgr;·<IT>K</IT> (ml·100 ml tissue<SUP>−1</SUP>·min<SUP>−1</SUP>)
where lambda  is the partition coefficient of tissue (adipose)/blood (in µBq g tissue-1/µBq ml blood-1), which was 3.7 × 104 (5), and K is the elimination rate constant for the monoexponential washout of 133Xe.

NIRS-ICG blood flow. For NIRS-ICG blood flow measurements in both calf muscle and Achilles peritendon regions, a NIRO 300 (Hamamatsu Photonics, Herrsching, Germany) spectrophotometer with dual-channel NIR laser diodes was utilized to determine the arterial ICG concentrate ([ICG]) after bolus injections. One set of emitting and receiving optodes was placed in the vertical plane over the gastrocnemius at the position of maximum circumference, with an optode separation distance of 4 cm. At this spacing, the penetration depth was ~2 cm, which corresponds to a tissue volume of ~16 ml encompassed in the light signal. For the peritendinous region, the probes were placed on the medial aspect of the ankle between the Achilles tendon and the medial maleolus, and the separation distance was also 4 cm. The NIRS probes for the peritendon region were positioned directly over the 133Xe depot site on the same side of the ankle (Fig. 1). A schematic representation of the ICG tracer procedure for blood flow measurements with NIRS is shown in Fig. 2. After venous bolus injection, the ICG bolus circulates to the right heart and lungs and emerges into the arterial circulation. Arterial blood is withdrawn by a pump, and the [ICG] is recorded by photodensitometry, whereas, downstream in the tissue microcirculation, ICG accumulation is detected by measuring light attenuation with NIRS. Changes in tissue [ICG] were determined by measuring light attenuation at wavelengths of 775, 813, 850, and 913 nm, analyzed with an algorithm incorporating the modified Beer-Lambert law: A = alpha  × c × d×B + G, where A is the measured light attenuation, alpha  is the wavelength-specific extinction coefficient for ICG (in mol/cm), c is the concentration of ICG in moles, d is the distance between the optodes over the tissue surface, B is a differential pathlength factor reflecting the adjustment in chromophore absorption due to the extended pathlength due to scatter in biological tissue (8, 35), and G is a constant reflecting light scatter (Hamamatsu Photonics).


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  		Fig. 2.   Schematic representation of the NIRS measurements of indocyanine green (ICG) in tissue after venous bolus infusion. From top left, an ICG bolus is injected into the venous circulation; it passes through the heart and lungs and into the arterial circulation and into the microcirculation. The NIRS optodes positioned over the tissue detect the ICG at several wavelengths, and, by use of specific extinction coefficients in a matrix operation, the ICG curve is isolated. The dotted circles represent the vessels from which the NIRS signal is detected. vol, Volume; OD 1-4, wavelengths 775, 813, 850, and 913 nm, respectively (OD = optical density).

Because the measured light attenuation in tissue results not only from the ICG present during passage of the fraction of the injected bolus but also from the oxy- and deoxyhemoglobin present, the ICG contribution to the light absorption signal was isolated by using a matrix operation (MATLAB) incorporating pathlength-specific extinction coefficients for each of the chromophores at each wavelength employed by the NIRS device (Hamamatsu Photonics). This procedure allows a clear separation of the light absorption attributed to hemoglobin + myoglobin and to ICG. With this procedure, the ICG curve recorded has essentially no influence on the hemoglobin + myoglobin signal.

When the amount of tracer and the blood flow in the injection artery are known, the blood flow in the tissue region monitored by NIRS can be estimated from the peak of the recorded dye curve. Alternatively, blood flow can be calculated as the ratio of tissue ICG accumulation to the arterial input function, which is the average [ICG] determined in the arterial blood over the same time period (Fig. 3). According to the Sapirstein principle (32), for any given time interval less than the time to reach peak tissue accumulation of tracer, the tissue receives the same fraction of the cardiac output as the fraction of the bolus received. Assuming that the central dilution volume is constant, that there is adequate mixture of ICG in the blood, and that the injection volume of ICG is accurately determined, then flow can be expressed by
Blood flow (ml·100 ml·min<SUP>−1</SUP>)=<FR><NU><IT>k</IT>·[ICG]<SUB>m</SUB>·<IT>t</IT></NU><DE><SUB>0</SUB>∫<SUP><IT>t</IT></SUP>[ICG]<SUB>a</SUB> d<IT>t</IT></DE></FR>
where k is a constant for the conversion of ICG in moles to grams per liter measured from in vitro blood phantoms; [ICG]m is the accumulation of ICG in tissue over time t expressed in micromoles; and 0int t [ICG]a dt is the time integral of the arterial [ICG] expressed as milligrams per liter.


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  		Fig. 3.   Representative tracings of ICG dye accumulation in calf muscle determined by NIRS (NIRS-ICG) at rest (A), and during plantar flexion exercise at 7 W (B) in response to venous bolus infusion. C and D: corresponding arterial ICG curves determined by photodensitometry. Muscle blood flow is determined from the ratio of muscle ICG concentration ([ICG]) accumulation to the mean arterial [ICG] over the same time interval (see text). Dashed lines represent time intervals for ratio calculations of blood flow.

If there is an even distribution of the tracer within the monitored tissue volume when the ICG curve reaches its maximum height, the measured tracer concentration will reflect the blood flow in that region. If the injected bolus is incompletely mixed with the blood, the vascular pathways taken by the tracer will not be representative for the blood flow distribution. In this study, adequate mixing was ensured by injecting the ICG bolus into the venous circulation, providing sufficient admixture during passage through the heart and lungs before entry into the arterial and tissue circulations, respectively.

To negate the potential influence of ICG recirculation on the calculation of flow, the ICG accumulation was determined over a time interval less than the first half of the tracer transit time. This method (32) eliminates the uncertainty of when the first and last part of the bolus passes the tissue, and therefore the venous outflow [ICG] in the NIRS signal is not required to determine blood flow. Two to three separate time points within the first half of the curve were used to calculate the blood flow, and the average value was taken as that representative of the tissue ICG accumulation (see Fig. 3).

Other conditions must be fulfilled in order for tracer model calculations to be valid. First, blood flow must be constant for the period of measurement. Second, there must be linearity and stability of the tracer dose response in tissue, and third, the indicator must not be metabolized or permanently retained in the tissue during the period of measurement (17). These requirements were addressed in the calculations in the model of the present study. First, blood flow was measured after 3 min of exercise, ensuring steady-state conditions. Second, for accurate determination of arterial [ICG] by photodensitometry, standard-curve blood ICG calibrations were made by using known [ICG] in accurately measured volumes of each subject's blood after each experiment. Thus the photodensitometer voltage deflection recordings were accurately converted to milligrams per liter of ICG, and a linear relationship exists between [ICG] and light absorption in human whole blood measured by photodensitometry (Waters, Rochester, MN). The linearity of the NIRS-determined ICG response in tissue was assessed by a dose-response procedure involving femoral arterial injections of varying ICG volumes (see below). Finally, regarding tissue retention and clearance of the tracer, ICG is bound to plasma proteins with no retention in extravascular tissue spaces. Also, there is no clearance of ICG by the liver during the first pass of the bolus in each blood flow measurement sequence. Upon reaching the hepatic circulation, ICG is removed from plasma by hepatic parenchymal cells at a rate of ~0.8 mg/min in healthy adults (28). Thus clearance of a single ICG bolus (5 mg) is complete within 6-8 min. Nonetheless, for all ICG blood flow measurements, background ICG was subtracted from the NIRS signal, and the photodensitometry computer was reset to zero.

Tissue ICG accumulation and kinetics. An independent analysis of ICG accumulation in tissue measured by NIRS was made in all subjects at each exercise load with repeated femoral arterial injections of varying ICG volumes from a calibrated syringe. The purpose of this procedure was to assess the linearity of the NIRS-ICG response in tissue and to analyze both the magnitude and the kinetics of tissue ICG accumulation as a function of workload. This procedure was performed independent of the venous injection procedures used to determine blood flow (see Protocol). The recorded light attenuation curves after arterial injection were converted to micrograms ICG in the matrix operation described above. ICG coefficients describing the ratio of peak tissue NIRS-ICG accumulation vs. ICG bolus volume were derived for each subject at each exercise load. Regression equations were then derived to express ICG coefficients relative to workload. The transit time function of the ICG curve was also analyzed from the arterial injection procedure. For each ICG curve, the dispersion time was calculated, operationally defined as the interval between 10 and 90% of the peak ICG curve. This parameter represents the time interval from the appearance of the ICG in tissue to when 50% of the bolus has accumulated in the tissue region (22).

Protocol. After subjects rested for 10 min in the seated position, baseline resting blood flow was measured in calf muscle and peritendon regions with NIRS by venous bolus ICG injection, and cardiac output was determined by photodensitometry. Femoral arterial blood was withdrawn by a Harvard pump through the photodensitometer, and 5 mg of ICG were rapidly injected into the arm vein, followed by a 5-ml flush of saline. The arterial ICG dye curve was recorded after circulation to the femoral artery, and soon thereafter the NIRS-ICG signals in calf muscle and peritendinous tissue were recorded. The measurements of cardiac output and NIRS-ICG blood flow were completed within ~1 min. NIRS-ICG blood flow was measured twice at rest, separated by a 5-min interval, and averaged. Simultaneously, blood flow in the peritendinous region was determined by 133Xe washout. Leg blood flow was not measured at rest with dye dilution because, with low absolute limb blood flows at rest, the recirculation (second pass) of dye is often superimposed on the first circulation, rendering quantification inaccuracies.

Subjects then began rhythmic dynamic plantar flexion exercise at 1 W (60 contractions/min; metronome paced) on an ergometer for a period of 5 min. After 3 min of exercise, NIRS-ICG blood flow in the calf and peritendon regions was measured, along with cardiac output, by venous bolus injection. Peritendinous flow was determined simultaneously by 133Xe washout over the last 3 min of exercise.

Subjects then rested for 10 min and then repeated the same load, during which arterial ICG injection was performed to determine leg blood flow by dye dilution and photodensitometry, as well as to determine the magnitude and kinetics of ICG accumulation in tissue by NIRS. These measurements also began after 3 min of exercise. The same procedures were repeated for the 3-, 5-, and 7-W loads. The final exercise bout consisted of a ramped bout starting at 5 W for 1 min, followed by 7 W for 2 min, and finally 9 W for 2 min. During this bout, all measurements were made in the last 2 min of exercise.

Data analysis. Values are presented as means ± SE. Differences in blood flow with exercise and between methods were analyzed by the Friedman test, and if found significant such differences were located by Wilcoxon's test. Differences were considered significant if P < 0.05. Comparisons of blood flow between methods were also evaluated as described by Bland (1), whereby the difference in blood flow between methods is plotted against the average flow, and the mean difference is determined along with limits of agreement based on the standard deviation of the differences.

Linear regression analysis was used to evaluate the dose-response calibrations of tissue ICG accumulation vs. arterial ICG bolus volume (NIRS-ICG coefficients), and rate constants for ICG dispersion times as a function of workload were derived.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Calf muscle blood flow: NIRS-ICG vs. dye dilution. Representative tracings of the arterial ICG response, determined by photodensitometry, and the ICG accumulation in calf muscle, measured by NIRS after venous bolus infusion of ICG, are shown in Fig. 3. Dynamic changes in both the arterial ICG input function and the tissue ICG accumulation occurred with exercise. The magnitude and rate of muscle ICG accumulation increased for a given bolus injection of ICG with increasing workload. In the artery, the time-integrated [ICG] decreased from rest to exercise for a given injection volume, reflecting an increase in arterial blood flow.

On the basis of the MRI scans, the muscle volume of the whole lower leg was 1,755 ± 91 ml, whereas the volume of the triceps surae muscles alone was 1,330 ± 69 ml, and that of the Achilles tendon was 80 ± 5 ml. Calf muscle blood flows measured by NIRS with venous ICG bolus injection are shown in Fig. 4 and compared with those determined by dye-dilution photodensitometry with the MRI muscle volume of the whole lower leg. At rest, calf muscle blood flow measured by NIRS with venous ICG injection was 2.4 ± 0.2 ml · 100 ml-1 · min-1. During exercise, calf blood flows (ml · 100 ml-1 · min-1) measured by NIRS vs. dye dilution, respectively, were 11.2 ± 1 vs. 15.6 ± 1 at 1 W, 23.7 ± 4 vs. 32.1 ± 4 at 3 W, 46.8 ± 6 vs. 51.5 ± 6 at 5 W, 61 ± 4 vs. 69.9 ± 5 at 7 W, and 74.7 ± 5 vs. 77.1 ± 6 at the peak load of 9 W. There were no differences in blood flow between methods (P < 0.05).


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  		Fig. 4.   Top: calf blood flow responses to incremental plantar flexion exercise determined by NIRS-ICG and reverse dye expressed per 100 ml on the basis of the muscle volume of the whole lower leg (1,754 ± 90 ml) measured by magnetic resonance imaging (MRI). Bottom: Achilles peritendinous blood flow determined by NIRS-ICG and 133Xe washout. There were no differences between the NIRS-ICG and standard blood flow values, P < 0.05.

In Fig. 5, calf muscle blood flow measured by NIRS with venous injection is compared with the blood flow determined by dye-dilution photodensitometry extrapolated to the volume of only the triceps surae muscles determined by MRI. The latter blood flow estimate follows the assumption that only the gastrocnemius and soleus muscles were activated during incremental plantar flexion exercise. With this muscle volume estimate, the dye-dilution blood flow was significantly higher at all workloads (P < 0.05).


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  		Fig. 5.   Comparison of calf muscle blood flow during plantar flexion exercise determined by NIRS-ICG and reverse dye (Rev-dye) combined with MRI assessment of the muscle volume of only the triceps surae muscles (volume 1,330 ± 69 ml). Blood flow measured by NIRS-ICG was consistently higher at all workloads compared with the dye-dilution method extrapolated to the volume of only the gastrocnemius and soleus muscles, P < 0.05.

The mean difference between the dye-dilution method using whole leg muscle volume and the NIRS-ICG method was 5.7 ml · 100 ml-1 · min-1, and the 95% confidence interval for the bias was -10 to +20 ml · 100 ml-1 · min-1 (Fig. 6). When only the triceps surae muscle volume was assumed, the mean difference between methods was 21 ml · 100 ml-1 · min-1, and the 95% confidence interval for the bias was -3 to +46 ml · 100 ml-1 · min-1.


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  		Fig. 6.   A: Bland and Altman plot showing differences between the blood flows measured by 133Xe-washout and NIRS-ICG methods for the Achilles peritendon region. The mean difference between methods was 0.41 ml · 100 ml-1 · min-1, and the 95% confidence interval is +5.4 to -5.1 ml · 100 ml-1 · min-1. Similar comparison for calf muscle show differences between the reverse-dye and NIRS-ICG methods incorporating the muscle volume of the whole lower leg (B; 1,754 ± 90 ml) and only the gastrocnemius and soleus volumes (C; 1,330 ± 69 ml). As shown, the reverse-dye method was, on average, 5.7 ml · 100 ml-1 · min-1 higher than the NIRS-ICG values based on the whole leg volume and 22 ml · 100 ml-1 · min-1 when only the triceps was assumed.

Peritendon blood flow: NIRS-ICG vs. 133Xe washout. The peritendon blood flows measured by NIRS with venous ICG injection were compared with those measured by 133Xe washout, and the results are also shown in Fig. 4. At rest, blood flow measured by NIRS was 2.2 ± 0.5, whereas that measured by 133Xe washout was 2.0 ± 0.5 ml · 100 ml-1 · min-1. During exercise, peritendon blood flows (ml · 100 ml-1 · min-1) measured by NIRS vs. 133Xe washout, respectively, were 4.6 ± 1 vs. 3.9 ± 0.8 at 1 W, 8.2 ± 1 vs. 8.4 ± 2 at 3 W, 10.8 ± 2 vs. 12.6 ± 3 at 5 W, 13.6 ± 1 vs. 14.6 ± 3 at 7 W, and 14.7 ± 1 vs. 13.2 ± 3 at 9 W. There were no differences in blood flow between methods (P < 0.05). The mean difference between the 133Xe washout and the NIRS-ICG methods compared across all subjects and exercise loads was 0.41 ml · 100 ml-1 · min-1, and the 95% confidence interval for the bias was -5.1 to +5.4 ml · 100 ml-1 · min-1 (Fig. 6).

NIRS-ICG coefficients and dispersion times. The results of the NIRS responses to arterial bolus infusion of varying ICG volumes are presented in Figs. 7-9. Figure 7 shows the NIRS-ICG response in calf muscle to different doses of ICG injected into the femoral artery during steady-state exercise. The similarity of the ICG coefficient (ICG accumulation/ICG dose) demonstrates linearity of the NIRS signal, i.e., doubling the ICG dose elicited a twofold greater tissue ICG accumulation at a given exercise load. In Fig. 8, the grouped mean ICG coefficients are plotted as a function of workload, and for both calf muscle and peritendon regions there was a linear increase in tissue ICG accumulation for a given ICG dose as workload increased. The slope of the regression line was less than unity in both tissue regions, but that for muscle was sixfold higher than in the peritendon region.


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  		Fig. 7.   Representative NIRS-ICG curves in response to femoral artery bolus infusion of ICG during exercise. Coefficients for ICG (mol ICG accumulated per mg infused; A, B) were determined to assess the linearity of the tracer response. As shown, doubling the ICG bolus volume results in a twofold greater accumulation of ICG in muscle and a constant ICG coefficient (B) at a given work rate.



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  		Fig. 8.   Mean ICG coefficients (Coef) determined from arterial bolus infusion for 5 subjects during incremental plantar flexion exercise. Coefficients for calf muscle (A) and for Achilles peritendinous tissue (B) are shown as a function of workload.



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  		Fig. 9.   ICG dispersion times in calf muscle and peritendon regions, representing the time interval for half of the ICG bolus to accumulate in the tissue. Dispersion kinetics change similarly in both tissues, but from rest to exercise, dispersion time decreased 10-fold, whereas decrease in the Achilles peritendon region was 3.3-fold. *Difference from prior exercise load; #differences between tissue regions; P < 0.05.

ICG dispersion times for calf muscle and Achilles peritendon regions are presented in Fig. 9. Mean dispersion times decreased exponentially in both tissues with increasing exercise load, and the rate constant in calf muscle was twofold greater than in the peritendon region.

Systemic and regional hemodynamics. At rest, cardiac output was 5.8 ± 0.2 l/min, and mean arterial blood pressure was 83 ± 1 mmHg (Fig. 10). During exercise, cardiac output increased to 9.5 ± 0.6 l/min at the peak load of 9 W, and mean arterial pressure increased to 116 ± 3 mmHg (P < 0.05). Accordingly, calf muscle and Achilles peritendon vascular conductances based on the NIRS-ICG blood flow increased to 0.64 ± 0.02 and 0.13 ± 0.02 ml · 100 ml-1 · min-1 mmHg-1 (P < 0.05), respectively (Fig. 10).


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  		Fig. 10.   Systemic and regional hemodynamics during exercise: combined blood flow responses in the lower leg measured by NIRS-ICG (A) and cardiac output (B), mean arterial pressure (C), and vascular conductances (D) in calf muscle and the peritendinous regions. *Difference from previous exercise load; #difference between tissues; P < 0.05.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present study is the first to quantify blood flow in muscle and other tissues in humans during exercise with NIRS using ICG dye. The results indicate that this method is sensitive for detecting progressive changes in tissue perfusion induced by incremental exercise and for assessing perfusion differences between tissue regions. The validity of the NIRS-ICG method was established by comparison with other established flow techniques in which good agreement was found. Thus NIRS may serve as a valuable tool for examining regional blood flow distribution and regulation during exercise in humans.

In this study, blood flow was quantified with NIRS using venous bolus injection of ICG. This is a simple procedure and is essentially the same as a cardiac output measurement by dye dilution. ICG is injected into a vein, and, once the dye circulates to the arterial circulation, the ICG curves are recorded from the artery by photodensitometry and in tissue by NIRS. Blood flow is computed as the ratio of tissue ICG accumulation to the mean arterial [ICG] over a specified time interval. In the present experimental setup, it is also possible to calculate blood flow with NIRS on the basis of the femoral arterial ICG infusion procedure. We did not elect this method because it is technically more difficult and, in our view, less accurate than the venous method. Because the arterial [ICG] is necessary in the model calculation of blood flow, the arterial infusion procedure requires a calculation of the instantaneous blood volume present in the femoral artery at the moment of ICG infusion. This can be derived from the whole leg blood flow, determined by dye-dilution photodensitometry, divided by the time interval of the bolus infusion, which needs to be measured. Also, our studies indicate that the timing of the infusion with respect to the cardiac cycle and muscle duty cycle may affect the estimation of the arterial [ICG]. Finally, in contrast to the venous injection method, which allows for several blood flow calculations from a single ICG curve, only one average arterial [ICG] is obtained with arterial infusion. These factors render the arterial infusion method more complex and less accurate for quantifying blood flow with NIRS.

Dynamics of ICG accumulation. Analysis of the ICG responses in the femoral artery as measured by photodensitometry and in the tissue as determined by NIRS indicate that both the arterial input function (time-integrated arterial [ICG]) and tissue ICG accumulation are important parameters for the calculation of blood flow. The mean arterial [ICG] is a function of both the unit blood volume present at the time of injection and the arterial flow rate, which affects dispersion of the arterial ICG curve. This is illustrated in Fig. 3, whereby both the magnitude of the peak concentration as well as the kinetics of the arterial ICG curve change as a function of exercise load. For a given ICG injection volume, the mean arterial [ICG] decreases as the arterial flow rate increases, and, accordingly, changes in arterial [ICG] contribute to the tissue blood flow calculation.

The arterial bolus injection experiments were used to determine blood flow across the lower leg by photodensitometry and to analyze the dose response and kinetics of ICG accumulation in tissue by NIRS with varied ICG infusion volumes. At a given exercise load, the ICG coefficients were identical with varying ICG infusion volumes, indicating linearity of the NIRS-ICG signal (Fig. 7). On the other hand, with increments in the workload there was a greater magnitude of ICG accumulation for a given volume infusion in both tissues. The ICG dispersion times shown in Fig. 6 also indicate that with increments in work rate there is a more rapid accumulation of ICG in tissue. Increases in both the rate and magnitude of ICG accumulation can be attributed to local vasodilation producing a more uniform perfusion in the vessels in the region and also to an elevated perfusion pressure. Given that both muscle and peritendon regions were influenced by a similar mean pressure at the level of the large feed arteries, the lower dispersion times and larger ICG coefficients in muscle compared with the peritendon region provide some indication of differential levels of vasomotor tone between these tissues. This is also illustrated by the pattern of vascular conductance in these tissues, indicating greater vasodilation and vascular recruitment in muscle compared with around the tendon (Fig. 10). Taken together, changes in the magnitude and kinetics of ICG accumulation in tissue reflect that increases in tissue blood flow during exercise result from a larger vascular recruitment and a more rapid blood velocity through tissue.

Comparison of blood flow methods. In the Achilles peritendon region, close agreement was established between the NIRS-ICG method and 133Xe washout (Figs. 7 and 9). The 133Xe washout method has been shown to be a valid method for quantifying blood flow in this tissue region (16). We avoided the use of 133Xe washout in calf muscle for comparisons to the NIRS-ICG method because numerous radioisotope injections would have been required due to its rapid washout in muscle and because there are systematic errors in 133Xe clearance from muscle (2, 13). Thus the NIRS-ICG blood flows were compared with the standard of dye dilution in combination with MRI-determined calf volume whereby whole leg blood flow was extrapolated to unit volume flow per 100 milliliters of tissue. Good agreement was found between methods for muscle when the dye dilution was extrapolated to the volume of the whole lower leg muscle. When only the triceps surae muscle volume was incorporated, the blood flow estimates were significantly higher than the NIRS values, and greater variability existed between the methods, as shown in Fig. 6. Although dye dilution is an established method for whole limb blood flow measurements, only a relative comparison can be made to the NIRS-ICG method because of the uncertainty of the actual muscle volume engaged during exercise (20, 29). Nonetheless, assuming that blood flow increased to the whole lower leg during dynamic plantar flexion, as shown by Frank et al. (11), the present results suggest good agreement between methods.

Perspectives. An important question in exercise physiology is how blood flow is distributed within and among specific tissues during exercise in humans. The difficulty in addressing this question with limb blood flow methodologies relates to the uncertainty in defining which regions of a large muscle group receive a given flow measured from a large limb artery or vein. By using the dynamic knee extension model together with thermodilution and ultrasound Doppler measurements of limb flow from the femoral vein and artery, respectively, it has been established that quadriceps muscle blood flow increases linearly with exercise load (31). However, this pattern may not exist for each muscle or for specific regions within each muscle. With NIRS used to measure oxygen saturation of different quadriceps muscles during knee extension exercise, heterogeneous saturation levels have been found between muscles and in different regions of the same muscle (10, 18). The extent to which these responses are coupled with blood flow heterogeneity is unknown.

Recent estimates of muscle recruitment and blood flow determined after knee extension exercise measured by transverse relaxation time-weighted MRI suggest that muscle volume recruitment progresses with incremental power output (26) and that flow increases are matched to defined units of activated muscle. It has been proposed that, even at submaximal exercise loads, near-maximal flow rates are reached within the specific regions of activated muscle and that, with incremental exercise loads, flow to the muscle group as a whole increases by the recruitment of additional muscle with its corresponding near-peak flow rate. This is an interesting hypothesis that necessitates further investigation.

Using arterial spin-labeling MRI, Frank et al. (11) have recently quantified regional perfusion in the muscles of the lower leg during graded plantar flexion exercise. Their high-spatial-resolution measurements confirmed that perfusion heterogeneity exists within regions of the same muscle and between the various muscles of the lower leg, and they also found differential increments in blood flow in the same muscle and between muscles with increasing exercise intensity. It is unclear whether their findings confirm the hypothesis of Ray and Dudley (26). Further work with these methods is needed to ascertain how blood flow is matched to muscle activation and to identify the mechanistic basis for regional blood flow distribution during exercise in humans.

Advantages and limitations of NIRS. Some advantages of the NIRS technique for measuring regional blood flow are its ease of application, good signal integrity during robust body movement such as exercise, its potential for temporal and spatial resolution, and the fact that the signal primarily reflects microcirculatory blood flow. Multiple NIRS channels would allow mapping of blood flow patterns within and between muscles and other tissues, which could be measured in close temporal proximity to other indexes of oxygenation. Blood flow kinetics such as transit time can be determined, and changes in hemoglobin volume in relation to regional blood flow may be applicable as an index of local vasomotor tone. These parameters could prove useful for studying microvascular control mechanisms. As in the experimental setup in this study, it is also possible to describe regional hemodynamic patterns in relation to systemic responses as shown in Fig. 10. For a given increase in cardiac output, the higher blood flow in muscle compared with that around tendon is associated with a larger vascular conductance, reflecting a differential level of vasomotor tone. Despite a substantial increase in cardiac output, blood flow around the tendon reaches only ~20% of its peak reactive hyperemic flow rate (3), therefore indicating tonic restraint of vasodilation.

Although the present blood flow technique offers several unique advantages for examining regional blood flow distribution during exercise, there are some limitations. The tissue volume estimate in the NIRS signal with an optode spacing of 4 cm used in this study is ~16 ml, which may be relatively large compared with the spatial resolution provided by other methods (11, 33). The volume of the vascular compartment of interest is, however, much smaller, i.e., in the range of 1-4 ml depending on the degree of vasodilation (C. A. Piantadosi, personal communication). The tissue volume estimate corresponds to the assumption that photons migrate through tissue in a spherical path, described generally by the equation volume = 2/3pi r3, where r is equal to one-half the optode spacing. This estimate may not be precise, especially between individual subjects. Further work with direct measurements of photon flight in tissue is needed to accurately quantify the absolute hemoglobin volume and thereby the vascular compartment volume during exercise.

Another potential limitation of NIRS at present is that, despite varying of the optode separation distance to allow for differential light penetration depths into tissue, for the most part, the signal reflects superficial regions of the monitored tissue. In addition, the gain in tissue penetration depth obtained by increasing optode spacing is offset by a loss of spatial resolution.

In summary, the results of the present study indicate that NIRS is a valid and sensitive method for quantifying regional tissue blood flow during exercise. The method has good temporal and spatial resolution, offers versatility for monitoring blood flow in a variety of tissues, and along with complementary indexes of oxygenation may provide insights into mechanisms regulating tissue oxygen delivery and utilization under varying conditions in health and disease.


    ACKNOWLEDGEMENTS

We thank Mark Cope for technical assistance.


    FOOTNOTES

This study was supported by the Danish National Research Foundation (Grant 504-14) and the Danish Medical Research Council (Grant 9802636).

Address for reprint requests and other correspondence: R. Boushel, Dept. of Exercise Science, Concordia Univ., 7141 Sherbrooke St. W., Montreal, Quebec, Canada H4B 1R6 (boushel{at}alcor.concordia.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 24 March 2000; accepted in final form 17 June 2000.


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