The use of near-infrared spectroscopy in understanding skeletal muscle physiology: recent developments

1. Introduction

Since the end of the 1980s, near-infrared spectroscopy (NIRS) has been used to investigate local muscle oxidative metabolism at rest and during different exercise modalities. The unique advantage of using NIRS is that, when proper care is taken to minimize movement artefacts, it can yield acceptable signal-to-noise ratios during exercise. Ferrari et al. [1] reviewed the first decade of NIRS muscle studies at the dedicated 1996 Royal Society Discussion Meeting. In the following years, several other reviews covered different aspects of muscle NIRS research such as sports science and clinical medicine [2,3]. Hamaoka et al. [4] reviewed the methodological issues of NIRS and near-infrared (NIR) imaging for monitoring muscle oxygenation and haemodynamics in healthy and diseased humans. The 234 references quoted in that review article witness not only the evolution of the NIRS/NIR imaging techniques, but also their significant applications in exercise physiology and clinical medicine.

A search on the databases PubMed, Scopus and Web of Science was performed using the keywords: ‘near-infrared’, ‘near-infrared oximetry’, ‘muscle oxygenation’ and ‘optical imaging’. From 2007 up to the end of 2010, approximately 160 articles related to muscle NIRS studies (excluding the clinical related studies) were published on different aspects of muscle physiology (primarily muscle oxygenation and haemoglobin (Hb) volume) of several upper and lower limb muscle groups investigated by using mainly one-/two-channel commercial NIRS instruments. Only 15 of these studies exploited the advantages linked to the use of multi-channel instruments.

Considering that the number of laboratories and articles related to the application of NIRS in muscle studies is increasing, most muscle physiology researchers would agree with the assertion that muscle NIRS research is progressing. The technology and methodology of NIRS instruments have shown advances in robustness and sophistication. This article is an attempt to provide a snapshot of muscle NIRS at the end of 2010 summarizing the recent literature, offering the present status and perspectives of the NIRS instrumentation and methods, the main fields of applications for studying skeletal muscle physiology, the open questions and future directions.

2. Present status and perspectives of the near-infrared spectroscopy muscle instrumentation and methods

(b) New methods and signal interpretability

The muscle parameters measurable directly and indirectly by NIRS and NIR imaging have been reviewed by Wolf et al. [5]. Muscle NIRS parameter measurements are repeatable and reproducible during elbow flexor exercise [8] and cycling exercise [9].

An example of the typical changes in biceps brachii SmO₂ and tHb (measured by a two-channel oximeter) during a 10 s sustained and 30 repeated isometric exercise of the elbow flexors at maximal voluntary contraction is shown in figure 1. During the sustained contraction (figure 1a), ΔSmO₂ desaturation slope (ΔSmO_2slope) is the negative slope of the least-squared regression line of ΔSmO₂ during the contraction phase; a higher ΔSmO_2slope would represent a greater muscle O₂ demand, and consequently a greater energy consumption [8,10]. ΔSmO₂ minimum amplitude (ΔSmO is the difference between the minimum SmO₂ value reached during the contraction phase and SmO₂ baseline; lower ΔSmO values represent greater O₂ demand relative to O₂ supply [6,10]. ΔSmO₂ maximum amplitude (ΔSmO is the difference between the maximum ΔSmO₂ value reached during the relaxation phase and SmO₂ baseline; higher ΔSmO_2max values indicate increased O₂ supply relative to O₂ demand [6]. ΔSmO₂ half-recovery time (ΔSmO_21/2RT) is the time to reach 50 per cent of the difference between ΔSmO₂ at the end of the contraction phase and ΔSmO_2max in the recovery period; a faster ΔSmO_21/2RT is related to muscle oxidative capacity [8]. Mean decrease in ΔtHb (ΔtHb_mean) is the difference between the average ΔtHb value during the contraction phase and tHb baseline; greater decreases and relatively stable ΔtHb_mean values during the contraction phase would indicate blood flow/O₂ supply occlusion owing to increased intramuscular pressure of the contraction [11]. Maximum increase in ΔtHb (ΔtHb is the difference between the maximum ΔtHb value reached during the relaxation phase and tHb baseline; higher ΔtHb values would indicate a greater increase in blood volume/flow [6].

• Download figure
• Open in new tab
• Download PowerPoint

During dynamic exercise (figure 1b), ΔSmO is the difference between the minimum ΔSmO value reached during the 30 contractions and SmO₂ baseline; a given ΔSmO value represents the dynamic balance of O₂ supply by the microcirculation and O₂ consumption by the mitochondria [8]. ΔSmO₂ desaturation time (ΔSmO_21/2DT) is the time difference between contraction onset until ΔSmO₂ reaches 50 per cent of the difference value between SmO₂ baseline and ΔSmO; a longer duration ΔSmO_21/2DT for a similar ΔSmO would represent a slower desaturation rate, indicating that O₂ demand is better matched by O₂ supply. ΔtHb_mean is the difference between the average of the minimum ΔtHb amplitude reached during the 30 contractions and tHb baseline; a given ΔtHb_mean value would indicate the level of blood volume or blood flow/O₂ supply over the exercise duration [11].

The kinetics of muscle oxygenation can be modelled by nonlinear regression using least-squares techniques [12], and the kinetics of SmO₂ is represented by the following formula:

where SmO_2base represents the resting SmO₂ baseline value. The curve fit for the first 30 s of exercise is shown in the inset of figure 1. After the onset of exercise (represented by the first dashed line), an initial time delay (SmO_2TD), followed by a rapid desaturation in SmO₂, is described with an exponential time constant (SmO₂τ) representing the time to reach 63 per cent of the desaturation response. SmO₂ desaturation amplitude (SmO_2A) is the difference between the SmO_2base and the nadir of SmO₂. The SmO₂ mean response time (SmO_2MRT = SmO_2TD + SmO₂τ) can be calculated to provide a description of the overall time course for muscle desaturation during the rest–exercise transition. A faster SmO_2MRT would indicate that O₂ consumption is not sufficiently being matched by O₂ supply, and conversely, a slower SmO_2MRT would suggest that O₂ consumption is sufficiently being matched by O₂ supply [12].

Many studies have evaluated the kinetics of muscle oxygenation using CW–SRS-based oximeters and have drawn their physiological conclusions only on the basis of the interpretation of the changes in HHb. Several arguments have been raised to support [13] or refute [14] this choice, because these oximeters provide SmO₂ values that are independent of the pathlength of the NIR photons in the muscle tissue and, unlike HHb, are not so sensitive to the optical coupling and to the presence of superficial tissue layers. Taking into account the advantages offered in using CW–SRS-based oximeters, the physiological conclusions should be drawn essentially on the basis of SmO₂ results, and reporting tHb data.

Among the most recent methods, Leung et al. [15] determined SmO₂ during cycling exercise measuring the rhythmical changes of the NIR signal or ‘cyclic SmO₂’. Binzoni et al. [7] further developed Leung's method by using the ‘cyclic’ signal embedded in the NIR signal to derive instantaneous SmO₂ and muscle O₂ consumption.

3. Main fields of near-infrared spectroscopy applications for studying skeletal muscle physiology

So far, only about 20 out of the approximately 600 skeletal muscles have been investigated by NIRS. Specifically, lower limb muscles (i.e. biceps femoris, gastrocnemius, rectus femoris, tibialis anterior, vastus lateralis, vastus medialis) were studied during diverse conditions (cycling, upon electrical stimulation, during knee extension exercise, leg press exercise, plantar flexion exercise, running, squatting, Wingate test), and upper limb muscles (i.e. biceps brachii, brachioradialis, deltoid, forearm flexors, triceps brachii) during diverse conditions (arm abduction exercise, bench press exercise, cycling, elbow flexion exercise, upon electrical stimulation, handgrip, rowing, Wingate test). The trunk muscles (i.e. erector spinae, intercostal, multifidus, paravertebral, serratus anterior) were also investigated during cycling, back extension and bending forward.

So far, NIRS has been applied for studying exercise-induced muscle damage [11,20], ergonomics/biomechanics [21], heterogeneity of muscle O₂ supply/demand [12,22], muscle activation [11,23], priming exercise [19,24], respiratory muscle blood flow/fatigue [25,26], the role of the brain in muscle fatigue [27,28], the time course of oxidative metabolism [6,29] and the effect of exercise training [30]. Owing to the restriction of the allocated space, this review article neglects an in-depth discussion of the 160 studies published in the last four years.

It is important to mention that among the muscle NIRS articles published in the last four years only 15 were performed by using NIR imagers. It is envisioned that more studies in the future will use the benefits of NIR imagers to study regional differences in muscle O₂ supply–demand responses to exercise. The effects of different motor tasks [31] and the effects of exercise/muscle fatigue [32] on both muscle and cortical oxygenation changes have been previously reviewed.

4. Open questions

Some of the most significant open questions in order of importance are: (i) the possibility to use NIRS in subjects with a large fat layer; (ii) the contribution of myoglobin (Mb) desaturation to the NIRS signal during exercise; (iii) the effect of scattering changes during exercise on CW–NIRS muscle measurements; (iv) the effect of changes in skin perfusion on CW–NIRS muscle measurements, particularly during prolonged exercise; and (v) the possibility to measure muscle/interstitial pH.

It is well known that the relatively high attenuation of NIR light in muscle measurements is owing to: (i) the two main chromophores—Hb and Mb; (ii) light scattering; and (iii) other molecules (mainly skin melanin, water, lipids of the adipose tissue, intra-muscle lipids and cytochrome c oxidase). The influence of adipose tissue thickness (ATT) on light propagation in leg muscles has been examined by several researchers. Muscle NIRS testing has been limited to slim subjects (preferably men because they have a smaller ATT with respect to women). Patients with diabetes, paraplegia or chronic fatigue syndrome, who have a tendency to be obese, cannot be investigated by NIRS if ATT values are high. At least three methods have been developed for fat-layer correction. An algorithm capable of correcting for the influence of ATT ranging from 0 to 15 mm has been proposed [33] and it has been included in some commercial NIRS systems (table 1). Another ATT correction algorithm and an optical method for ATT monitoring have been developed [34,35]. The optical lipid signal is a good predictor for ATT less than 16 mm. The possibility of measuring ATT directly might be an efficient alternative to the measurement of ATT by ultrasound. Another method for measuring SmO₂ has been recently reported [36]. This method uses broadband CW–NIRS after the removal of spectral interference owing to skin, water, ATT and scattering. Unfortunately, none of these methods have been so far used by the most widespread commercial NIRS system manufacturers (table 1), which strongly limits the clinical applicability of muscle NIRS in patients with high ATT.

There is a considerable discussion about the contribution of Hb and Mb to the in vivo NIR signal from skeletal muscle. It is difficult to differentiate Hb and Mb spectra because they are very similar in the NIR range. Marcinek et al. [37], using wavelength shift analysis, showed that Hb accounts for approximately 20 per cent of the optical signal in human resting first dorsal interosseous muscle. However, Hb/Mb ratio in the muscle at rest is unknown, and there are few data available on the relative kinetics of Mb and Hb desaturation during different exercise modalities. In an animal study, Mb desaturation of rat gastrocnemious muscle was investigated by NIRS during Hb-free medium perfusion [38]. At the onset of contraction, Mb desaturated rapidly and declined progressively with work intensity. Proton (¹H) NMR measures muscle deoxy-Mb signal allowing the assessment of intracellular O₂ availability at rest and during exercise with a time resolution of about 8 s. In a human study, Lanza et al. [39] found that, at rest, the tibialis anterior intramuscular O₂ stores (measured by the appearance of ¹H NMR deoxy-Mb signal during cuff occlusion) began to decrease only after 1 min, and that maximal Mb desaturation was achieved after about 6.5 min. Conversely, at rest, the intramuscular O₂ stores (measured by NIRS during cuff occlusion) in different muscle groups began to decrease a few seconds after the beginning of the occlusion and maximal desaturation was achieved 4–5 min later. During high-intensity exercise, Mb typically desaturates to only 50 per cent of the level attained during cuff occlusion, and muscle oxygenation, as measured by CW–NIRS, typically desaturates to about 90 per cent of the level attained during the cuff occlusion. Overall, these data would suggest that the contribution of Mb desaturation to the NIR signal during dynamic exercise is estimated to be roughly less than 20 per cent. Combined ¹H NMR and NIRS studies could clarify not only the issue of the contribution of Mb to the NIRS signal, but also the relative kinetics of Mb and Hb desaturation during exercise with different workloads.

In principle, exercise could alter the muscle tissue optical properties by causing a change in the scattering of NIR photons. The change in scattering at different wavelengths and the relative pathlength changes that occur during different exercise modes in the human quadriceps and calf muscles have been studied by TD–NIRS techniques [40,41]. It was observed that scattering coefficients and pathlength in the muscle, at different wavelengths, decreased by less than 10 per cent during the different exercise modalities. Therefore, it could be expected that scattering and pathlength changes, occurring during exercise, would negligibly contribute to the calculation of changes in O₂Hb, HHb and tHb measured by CW–NIRS instruments.

Several controversial studies have been published on the effect of skin blood flow changes on muscle NIRS measurements. A recent study [42] provided reassurance that changes in SmO₂ during exercise predominantly reflect muscle oxygenation, even during conditions where skin and muscle blood flow were elevated concomitantly (e.g. during prolonged, dynamic exercise).

Soller et al. [43] and previous studies showed that broadband CW–NIRS can be used to non-invasively and continuously measure SmO₂ (based on Hb spectral features), and indirectly muscle/interstitial fluid pH. Partial least-squares regression was used to develop the multi-subject calibration equation, which correlates NIR spectra to muscle/interstitial fluid pH values obtained invasively in the muscle of the calibration subjects. Once developed, the equation can be applied to NIR spectra acquired from independent subjects to predict muscle/interstitial pH. Recent studies demonstrated the feasibility of the muscle pH estimation, the determination of H⁺ threshold and the relation between H⁺ and classical metabolic thresholds (lactate and gas exchange) during incremental exercise [44].

A summary of the recommendations for properly using NIRS instrumentation in muscle studies is reported in table 2.

5. Future directions

The most exciting prospect of muscle NIRS studies for the next 20 years is the full understanding of skeletal muscle biochemistry/physiology/pathology for improving human healthcare, athletic performance and rehabilitation monitoring. The major challenge to achieving this understanding might be the availability of a low-cost, easy-use optical wearable/wireless non-contact NIR imager for obtaining four-dimensional SmO₂ and haemodynamic (blood flow and tHb) measurements of human skeletal muscle, especially during dynamic exercise. This ideal ‘NIR imager’ should be suitable for any application (including general health, clinical and athletic settings), and might be an addition to the current heart rate monitoring and lactate measurements during training in the field and health centre-like environments. This ideal ‘NIR imager’ should be combined/integrated with other imaging and electrophysiological modalities for enhancing the understanding of specific muscle mechanisms in pathophysiological conditions.

Considering the rapid development of related technologies, it is very difficult to predict the potential advancements of muscle NIRS and NIR imaging. The quantitative measurement of deep forearm oxygenation and tHb by a non-contact oximeter prototype was proposed by Niwayama et al. [45].

The current typical depth sensitivity of most CW-based imagers is approximately 1.5 cm. Therefore, a tomographic approach might provide three-dimensional SmO₂ and haemodynamic measurements. Blood flow of the superficial muscles might be continuously measured by diffusing-wave spectroscopy, a new rapidly progressing technique discussed in an accompanying symposium paper. Although three-dimensional NIR imaging of the human forearm, based on TD techniques, was proposed by Hillman et al. [46] almost 10 years ago, no further progress has been made to develop the technique.

In conclusion, it is foreseeable that the availability of advanced NIR imagers would help to refine the understanding of skeletal muscle oxygenation in different pathophysiological conditions.