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Near-infrared spectroscopy and skeletal muscle oxidative function in vivo in health and disease: a review from an exercise physiology perspective

Near-infrared spectroscopy and skeletal muscle oxidative function in vivo in health and disease:... In most daily activities related to work or leisure, the energy for muscle work substantially comes from oxidative metabolism. Functional limitations or impairments of this metabolism can significantly affect exercise toleran and performan. As a method for the functional evaluation of skeletal muscle oxidative metabolism, near-infrared spectroscopy (NIRS) has important strengths but also several limitations, some of which have been overcome by rent technological developments. Skeletal muscle fractional O2 extraction, the main variable which can be noninvasively evaluated by NIRS, is the result of the dynamic balan between O2 utilization and O2 delivery; it can yield relevant information on key physiological and pathophysiological mechanisms, relevant in the evaluation of exercise performan and exercise toleran in healthy subjects (in normal and in altered environmental conditions) and in patients. In the right hands, NIRS can offer insights into the physiological and pathophysiological adaptations to conditions of increased O2 needs that involve, in an integrated manner, different organs and systems of the body. In terms of patient evaluation, NIRS allows determination of the evolution of the functional impairments, to identify their correlations with clinical symptoms, to evaluate the effects of therapeutic or rehabilitative interventions, and to gain pathophysiological and diagnostic insights. © 2016 Society of Photo-Optical Instrumentation Engineers (SPIE) [DOI: 10.1117/1.JBO.21.9.091313] Keywords: near-infrared spectroscopy; skeletal muscle bioenergetics; skeletal muscle oxidative metabolism; exercise physiology; exercise toleran; review. Paper 150870SSVR reived Jan. 6, 2016; acpted for publication Jun. 28, 2016; published online Jul. 21, 2016. Introduction Near-infrared (NIR) spectroscopy (NIRS) applied to skeletal muscle has often been seen with some skepticism by many exercise physiologists. On at a scientific meeting, one of the authors of the present review was told by Dr. Bengt Saltin (one of the most influential exercise physiologists of the last decades): "Unless you manage to measure O2 consumption, this technique has not much physiological interest." We respectfully disagree with Dr. Saltin's statement. Although we recognize that NIRS has several limitations, some of which have been overcome by rent technological developments, the method also has important strengths and can give valuable (and noninvasive) functional insights into skeletal muscle oxidative metabolism in vivo during exercise, in health and disease. At this purpose, the present review has been devoted, from an "exercise physiology point of view," to discuss some of the main issues related with the role of NIRS in the functional assessment of oxidative metabolism in skeletal muscles during exercise, with specific attention to integrative aspects and to the factors limiting exercise toleran. Attention will also be paid to studies carried out in diseased populations, as well as to some rent and exciting technical and methodological developments. Oxidative Metabolism During Exercise and Tools of Investigation *Address corresponden to: Bruno Grassi, E-mail: bruno.grassi@uniud.it During supramaximal exercise lasting up to a few seconds, the metabolic power output in humans can increase by about 150 to 200 times compared with the value observed at rest. Among the tissues of the body, only skeletal muscle can sustain such extraordinary increases in metabolic power output, which is made possible by the splitting of high energy phosphates present in muscle as adenosine triphosphate (ATP) and creatine phosphate (PCr). If the exercise is carried out for longer periods of time (up to several minutes, or even hours), the maximal metabolic power output, which can be sustained, decreases hyperbolically as a function of the duration of the exercise1­4 and oxidative metabolism becomes the prevalent, or substantially the only, mechanism responsible for ATP resynthesis. In other words, in most of our daily activities, either related to work or leisure, the energy for muscle work substantially comes from the oxidation of glucose and lipids in muscle fibers, culminating in oxidative phosphorylation at the mitochondrial respiratory chain. It is not surprising, then, that the maximal power by oxidative metabolism and the fraction of this power, which can be sustained for relatively long periods of time, are intrinsically related to exercise performan and toleran. An impairment of September 2016 Journal of Biomedical Optics 091313-1 Vol. 21(9) Grassi and Quaresima: Near-infrared spectroscopy and skeletal muscle oxidative function. . . oxidative metabolism, which occurs in several types of patients, inevitably leads to a redud exercise toleran, which may represent one of the main determinants of the clinical picture and quality of life, as well as an important predictor of mortality.5 The availability of reliable tools to investigate in vivo, with precision and reproducibility, muscle oxidative metabolism, possibly noninvasively and with a reasonably elevated temporal resolution, appears therefore of utmost importan. This applies to athletes (sports activities), healthy subjects (both in normal and in altered environmental conditions), and patients affected by many diseases. In the patients, these tools would allow the follow-up of the impairments with time, the identification of potential correlations with clinical symptoms, the evaluation of the effects of therapeutic or rehabilitative interventions, and could also yield pathophysiological and diagnostic insights. _ Whole body oxygen consumption (VO2 ) can be measured with relatively good precision (variability of measurements of about 5% in the best laboratories) at the mouth of the subject _ _ or at the alveolar level6 (pulmonary VO2, VO2p ) by the classic open circuit method. These measurements have been carried out in innumerable studies and lead to the definition of conpts like _ the maximal aerobic power (VO2p;max ), which evaluates the integrated performan of the respiratory, cardiovascular, and skeletal muscle components of the O2 pathway from ambient air to the mitochondria of skeletal muscles. The availability of _ methods to determine VO2p with an elevated temporal resolution ("breath-by-breath" measurements)6­8 expanded the spectrum of the functional evaluation variables and allowed the introduction of conpts such as the "gas exchange threshold,"9 the "critical _ power,"1 or the various phases of the VO2 "kinetics."3,4,6,10 The _ main limitation of the whole body VO2p measurements resides in the impossibility to discriminate between the exercising muscles and the rest of the body, as well as between different muscles engaged in the exercise. Moreover, the presen of O2 stores between the site of measurement (the mouth or the alveoli) and the sites of gas exchange at the skeletal muscle level complicates data interpretation during metabolic transitions.6 A further limitation derives from the diffusion of automated and apparently easy-to-use "metabolimeters," which to the proliferation of data and studies of dubious quality. _ The relationship between VO2 , blood flow and O2 extraction can be expressed by the Fick equation, based on the principle of mass conservation. If applied "across" a single muscle or a single muscle group, the equation reads as follows: EQ-TARGET;temp:intralink-;e001;63;258 from the exercising muscle, with a resulting "contamination" of venous blood coming from other muscles. Alternative methods allowing a functional evaluation in vivo of skeletal muscle oxidative metabolism derive from the utilizations of phosphorus (31 P) or proton (1 H) nuclear magnetic resonan spectroscopy (31 P-MRS, and 1 H-MRS, respectively). As far as 31 P-MRS, the method has been utilized to determine the kinetics of recovery of muscle [PCr] following exercise. After assuming the equilibrium of the creatine kinase (CK) reaction, the rate of recovery of [PCr] is a function of mitochondrial ATP production, and therefore it can be considered a tool for evaluating the function of skeletal muscle oxidative metabolism.15 1 H-MRS, on the other hand, detects myoglobin (Mb) desaturation, thereby allowing an estimation of intrallular PO2.16,17 These technologically formidable methods are intrinsically limited by the very high cost of the instrumentation and by the strict constraints on the exercise modality determined by the size of the bore of the magnet. In short, the above mentioned methods, although widely utilized, have intrinsic limitations. Therefore, noninvasive, precise, reproducible, and relatively low-cost functional evaluation tools to be utilized in vivo, characterized by an elevated temporal resolution, allowing the discrimination between (and possibly within) skeletal muscle groups, would be needed. Does NIRS satisfy, at least in part, these needs? The following sections of the present review will deal with this issue. Near-Infrared Spectroscopy from an Exercise Physiology Point of View _ _ VO2m ¼ Qm à ½Cða-vÞO2m ; (1) _ _ _ in which VO2m represents muscle VO2 , Qm muscle blood flow, and Cða-vÞO2m arterial-venous O2 conntration differen across the muscle. By rearranging Eq. (1): EQ-TARGET;temp:intralink-;e002;63;181 Several exllent review articles about the application of NIRS in skeletal muscle have been published.18­25 These reviews carry the relevant information on principles, methods, instruments, and so on. Ferrari et al.24 included a useful list of practical recommendations. The present review article has been specifically devoted to discuss, from an "exercise physiology point of view," some of the main issues related with the role of NIRS in the functional evaluation in vivo of oxidative metabolism in skeletal muscles, within an integrated perspective related to exercise toleran. A specific attention will be paid to NIRS studies carried out in diseased populations, as well as to some rent and exciting technical and methodological developments. The latter testify how NIRS, as a tool of functional investigation of skeletal muscles, is still in a steep portion of a gain in knowge versus time curve. The role of NIRS in evaluating oxidative skeletal muscle performan in sport activities and in athletes, which would deserve a dedicated review article, is not covered in the present article. Likewise, the effects of exercise on brain hemodynamics, as measured by NIRS,26,27 as well as the role of NIRS in the evaluation of brain function21,25 are not discussed. Cða-vÞO2m _ _ ¼ VO2m Qm : (2) Tissue Interrogated by the Near-Infrared Spectroscopy Probe _ VO2m measurements have been carried out with a high temporal resolution also during metabolic transitions in humans11,12 or in isolated in situ animal models.13,14 The main limitations of this approach can be summarized as follows: invasiveness of the measurements; technical problems inherently associated with in vivo blood flow measurements; the substantial impossibility (in human studies) to sample venous blood directly effluent Journal of Biomedical Optics When the probe is applied on the skin overlying a muscle of interest, NIRS instruments can interrogate only a relatively small (2 to 6 cm3 ) and superficial volume of skeletal muscle tissue.28 It is generally acpted that the depth of penetration of the NIR light in tissues roughly corresponds to half of the distan between the light sour and the detector (which is usually between 3 and 5 cm). This intrinsic technical limitation makes the measurement of the thickness of the skin and September 2016 091313-2 Vol. 21(9) Grassi and Quaresima: Near-infrared spectroscopy and skeletal muscle oxidative function. . . subcutaneous adipose tissue layer, at the site of plament of the NIRS probe, mandatory. The measurement can be carried out by a caliper or, more precisely, by ultrasound.29,30 The rent availability of low-cost hand-held ultrasound devis has significantly facilitated these measurements. Although no cutoff values are recognized as standard, a value greater than 20 mm would presumably make NIRS measurements, as carried out by standard instruments, rather meaningless in terms of investigating skeletal muscle. This precludes the utilization of the method in subjects with a relatively thick layer of subcutaneous fat, such as obese patients or in patients with significant muscle atrophy. Table 1 Main commercial muscle NIRS oximeters. Devi Oxymon Mk IIIa Company, country Artinis, The Netherlands Astem, Japan Technique Multidistan CW Multidistan CW Multidistan CW Type of sours N. of wl 2 Timeresolution No. of Measurable (Hz) channels parameters Approvals 250 2 Websites www.artinis.com Exercise Monitor Hb11b www.astem-jp.com Fore-sight Elite®a CAS, Inc., United States NIRO-200 NXa , www.casmed.com Hamamatsu, Multidistan Japan CW Hamamatsu, TD Japan ISS, United States Masimo, United States Multidistan FD Multidistan CW www.hamamatsu.com tNIRS-1a , atHb www.hamamatsu.com OxiplexTSTMa , atHb www.iss.com O3TM Regional Oximetrya up to 6 , SO2p www.masimo.com INVOSTM 5100Ca Medtronic, United States moorVMS-NIRS Multidistan CW www.medtronic.com Moor, United Multidistan Kingdom CW Nirox, Italy Multidistan CW Multidistan CW www.moor.co.uk NIMOab www.nirox.it SenSmartTM Model X-100a Nonin, United States Omegawave, Japan Ornim, Inc., United States up to 5 , SO2p www.nonin.com BOM-L1 TR SF Single distan www.omegawave.co.jp roxTM 3210Fa Multidistan CW , flowc www.ornim.com CareGuideTM 4100 Reflectan, Multidistan Med., United CW States Multiple , pHm www.reflectanmedical. com Note: atHb, absolute total hemoglobin conntration; , European conformity marking as a medical devi; CW, continuous-wave spectroscopy; FD, frequency-domain spectroscopy; , Food and Drug Administration's approval; , light emitting diodes; N., number; pHm, muscle pH; , muscle oxyhemoglobin saturation; SO2p , peripheral arterial oxyhemoglobin saturation (measured by pulse oximetry); TD, time-domain spectroscopy; wl, wavelengths. a Brain oximeter utilized also in muscle studies b Oximeter with fat-layer compensation c Cortical microcirculation blood flow measurement using Doppler shift in coherent light signals. Journal of Biomedical Optics 091313-3 September 2016 Vol. 21(9) Grassi and Quaresima: Near-infrared spectroscopy and skeletal muscle oxidative function. . . The fact that the NIR light has to cross the skin and subcutaneous fat in order to reach the underlying skeletal muscle tissue inevitably causes a sensitivity problem and represents a "contamination" of the signal of interest, which is the one coming from skeletal muscle. The problem can be particularly significant in conditions in which skin blood flow increases during exercise for thermoregulatory purposes (see also below). Several "correction algorithms" have been proposed (see the discussion in Ferrari et al.24), which, however, are not included in most of the commercial instruments (Tables 1 and 2). Ohmae et al.31 have rently described a simple method, which does not require ultrasound, for the sensitivity correction for the influen of fat thickness on muscle oxygenation, based on the estimation of fat thickness by time-domain NIRS (TD-NIRS) (see below). van Beekvelt et al.32 and Koga et al.29 proposed other correction _ algorithms based on the assumption that resting VO2m [estimated on the basis of the conntration increases in deoxy-hemoglobin (Hb) and deoxy-myoglobin (Mb), [deoxy(Hb+Mb)]) after applying a cuff occluding the arterial circulation, see below] would be inversely correlated with skin + subcutaneous tissue thickness. Another approach, based on [total(Hb+Mb)] values obtained at rest by quantitative multichannel TD-NIRS, has been proposed.33 In the quadrips muscle (often investigated by NIRS studies) and in other muscles as well, deeper muscle regions are characterized by a greater proportion of oxidative fibers compared to superficial regions34 and by a more oxidative energy metabolism. Accordingly, the deeper fibers have a greater sensitivity toward vasodilatory control mechanisms.35 An anatomical "gradient" in fiber type is presumably associated with a gradient also in terms of metabolism and blood perfusion.36 This has been substantially confirmed, in the quadrips muscle of humans, in studies carried out by utilizing a "high-power" TD-NIRS instrument,37,38 which allows interrogation of slightly deeper portions of muscle. This instrument, which delivers NIR light with a power about 30 times higher than that of traditional NIRS instruments, allows an effective mean penetration depth of about 3 cm and may represent a substantial technical improvement. Another problem related to the small volume of tissue interrogated by conventional NIRS instruments derives from the fact that muscle activation and metabolism,39 and even more markedly so blood perfusion36,40,41 are heterogeneously distributed within and between exercising muscles. Exllent reviews on metabolic and blood flow heterogeneities in skeletal muscle, in health and disease, have been rently published.42,43 Sin skeletal muscle fractional O2 extraction (see below) can increase during exercise only three to four times above the values at rest, _ whereas VO2m can increase 100-fold, directing blood flow according to O2 needs is crucial to allow sustained contractile performan.43 Most studies (but not all44) found good matching between _ _ muscle O2 delivery (QO2m ¼ Qm à CaO2 , in which CaO2 rep_ resents the arterial O2 content), or substrates delivery, and VO2m between macroscopic (volume of several cm3 ) regions of a muscle, or between different muscles involved in the exercise.30,45,46 However, when the resolution capacity of the measurements went down to about 1 cm3 , the spatial matching _ _ between QO2m and VO2m (assessed via PCr depletion) was far from being perfect.45 The presen (or absen) of a matching between the two variables could be more functionally relevant at a microscopic level. According to Segal,47 the recruitment of contiguous "microvascular units," whose volume is orders of magnitude smaller than the volume investigated by NIRS, could be difficult to match with the recruitment of motor units, whose fibers are sparse within the muscle. In other words, blood flow cannot specifically increase to a specific muscle fiber or to the fibers of a motor unit,47 but must rather Table 2 Main commercial continuous wave portable/wearable NIRS systems for muscle studies with wireless data transmission. Devi OctaMon Company, country Artinis, The Netherlands Technique Singistan Multidistan n.a. N. of wl 2 Timeresolution (Hz) 50 No. of channels 8 Measurable parameters O2 HbHHb changes , LT Websites www.artinis.com PortaMon Mk IIb BSX Insight 2.0 Artinis, The Netherlands BSX Athletics, LLC, United States DynaSense Inc., Japan 2 n.a. 10 n.a. www.artinis.com www.bsxinsight.com PocketNIRS Duo®c,d Moxye Singistan Monte Carlo modeling Multidistan Multidistan O2 HbHHb changes www.dynasense.co.jp Moxy Monitor, United States Nirox, Italy Pathonix Innovation Inc., Canada www.moxymonitor. com www.nirox.it www.pathonix.com MyNimo OxiTor-M2 , atHb Note: atHb, absolute total hemoglobin conntration; HHb, deoxyhemoglobin; LT, lactate threshold evaluation by tracing; n.a., not available; N., number; O2 Hb, oxyhemoglobin; , muscle oxyhemoglobin saturation; wl, wavelengths. a Muscle imager. b Aclerometer is available on request. c Commercially available only in Japan. d Smartphone controllable system. e Water resistant case. Journal of Biomedical Optics 091313-4 September 2016 Vol. 21(9) Grassi and Quaresima: Near-infrared spectroscopy and skeletal muscle oxidative function. . . increase over a relatively wide region of the muscle, leading to the possibility of "over-perfusing" inactive or relatively inactive fibers. _ In any case, the apparently good matching between QO2m _ and VO2m in different macroscopic areas of the same muscle, or of different muscles,45,46,48 appears to be difficult to reconcile with the macroscopic heterogeneity of muscle deoxygenation observed by several studies.29,37,49 The issue, in our opinion, is not sett, and further studies with higher spatial resolution devis are needed. In summary, the area of muscle investigated by the NIRS probe may not represent, in terms of fiber types, fiber activation : _ and the matching of VO2m and QO2m , a reliable picture of the situation in the whole muscle. In this respect, however, it should be considered that the same limitation, frequently overlooked, is intrinsically associated with other diffusely utilized methods, which investigate only a small and relatively superficial portion of a muscle, such as muscle biopsy. Where do the Near-Infrared Spectroscopy Signals Come From? The NIRS signals are the result of the weighted average of the O2 saturations of the heme groups of Hb in the vascular bed (small arteries, arterioles, capillaries, venules, small veins) and of the Mb heme group in muscle fibers. In terms of Hb, most of the signal comes from small vessels, because larger arteries and veins (greater than 1 mm in diameter) have very high heme conntrations, which absorb all the NIR light. Although it is often considered that venous­venular compartments represent the majority of blood present in skeletal muscle,19,20 direct measurements suggest that this may not be the case. According to Poole and Mathieu-Costello,50 capillaries would contribute to >90% of the total blood volume in muscle. According to these data, the storage of blood in veins would mainly occur externally to the muscle. Thus, the vascular component of the NIRS signals would predominantly come from the capillaries. Sin in normal conditions, all regions of the muscle reive nearly-fully oxygenated arterial blood, oxygenation changes detected by NIRS would mainly reflect changes in capillary (Hb-related) and intrallular (Mb-related) O2 levels. A relative controversy exists about the role played in NIRS signals by the heme group in Mb. It has been traditionally assumed that Hb is responsible for most of the overall NIRS signal.51,52 The conpt is supported by the observation of strong correlations, observed in different experimental models, between muscle oxygenation values obtained by NIRS and O2 saturation in venous blood (see below). Studies carried out by NIRS in combination with 1 H-MRS, aimed at distinguishing between Hb and Mb desaturation during ischemia and exercise, yielded conflicting results.53,54 According to modeling papers,55,56 Mb may contribute to 50% of the total [Hb+Mb] NIRS signal. This conpt is supported by a study in which the contribution of Hb and Mb to in vivo optical spectra was determined by wavelength shift analysis.57 The relative role of Hb versus Mb is likely different at rest and during exercise. As discussed above, most of the intravascular NIRS signals likely come from the capillaries. In resting conditions, capillary hematocrit can be significantly lower than that in the systemic circulation.58 During exercise, on the other hand, capillary hematocrit increases, reaching values that are not substantially different from those in larger vessels.58 Thus, the Journal of Biomedical Optics role of Hb (versus that of Mb) in determining the NIRS signals would be higher during exercise compared to rest. The possibility to separate Hb and Mb desaturation could give valuable insights into the mechanisms regulating peripheral _ O2 diffusion and VO2m . For example, by applying NIRS on a rat model of isolated hindlimb, perfused with Hb-free welloxygenated Krebs-Henseleit buffer, Takakura et al.59 isolated the signal deriving from Mb desaturation and could calculate the intrallular PO2, making inferens on the role of Mb in O2 supply to mitochondria at exercise onset. In any case, the whole diffusion pathway, including vascular Hb and intrallular Mb, would desaturate during exercise with a similar time course.56 Therefore, a greater contribution of Mb than Hb to the NIRS [deoxy(Hb+Mb)] signal would not invalidate the interpretation that changes in this signal reflect frac_ tional O2 extraction, the dynamic balan between VO2m and _ QO2m in the volume of tissue under consideration.56 This would apply even if a separation between the Hb and the Mb signal is not feasible or is not carried out. In the next sections of the manuscript, we will discuss why, in our opinion, fractional O2 extraction is by itself of interest. In terms of the intrallular signals detectable by NIRS, cytochrome oxidase can also absorb NIR light. However, there is substantial agreement that assessment of the redox state of mitochondrial cytochrome oxidase cannot be done in muscle because of Mb interferen,60 whereas it is possible in the brain cortex.61 Which Variable Should be Considered? Upon the premise that NIRS oxygenation signals reflect fractional O2 extraction (see below) in the investigated volume of tissue, the following question can be asked: is there a correlation between NIRS-derived oxygenation signals and PO2 or Hb saturation in venous blood draining from the exercising muscle(s)? The answer to this question may not be straightforward, and conflicting results have been reported in the past. The issue is complicated by the problem of obtaining, particularly in humans, adequate samples of venous blood coming directly from the exercising muscle(s), without a "contamination" from blood coming from other muscles and/or other tissues. Wilson et al.62 found an exllent correlation between a NIRS oxygenation index and O2 saturation determined in the venous blood draining from a dog gracilis muscle preparation. The data were subsequently confirmed in humans during forearm exercise.53 On the other hand, in two studies63,64 performed during constant work rate exercise on a cycle ergometer, the following phenomena were observed (in normoxia): a transient decrease in oxygenation following the transition to exercise, which was paralle by a decrease in femoral vein O2 saturation; these decreases were followed by a paradoxical muscle "reoxygenation," which was not associated with an increased O2 saturation in the femoral vein. The latter, as expected, remained low for the remaining portion of exercise. In other words, following exercise onset an association between the two variables (decreased muscle oxygenation, decreased femoral vein O2 saturation) was observed, whereas dissociation occurred during the remaining portion of the constant work rate exercise. A similar muscle oxygenation pattern has been observed in different muscles14,65,66 for the oxygenated-Hb and -Mb ([oxy(Hb+Mb)]) signal (see below) while not for [deoxy(Hb+Mb)], which increased and then stayed constantly elevated during the remaining portion of the exercise. September 2016 091313-5 Vol. 21(9) Grassi and Quaresima: Near-infrared spectroscopy and skeletal muscle oxidative function. . . Grassi et al.65 reasoned that the muscle reoxygenation suggested by the [oxy(Hb+Mb)] time-course could derive from an increased blood flow to the skin, occurring for thermoregulatory purposes, as also suggested by the results obtained by Maehara et al.,67 Chuang et al.,68 and Davis et al.69 An increased skin blood flow for heat dispersion would increase the [oxy(Hb+ Mb)] signal, whereas would not substantially affect [deoxy (Hb+Mb)], sin the enhand blood flow would not be associated with an increased gas exchange. This was substantially confirmed in a study carried out by Koga et al.37 These authors observed by continuous-wave NIRS (CW-NIRS) (see below and Ferrari et al.24 for a definition), during whole body heating, a more pronound increase of [oxy(Hb+Mb)] compared to that of [deoxy(Hb+Mb)]. The apparent increased oxygenation described by CW-NIRS was not observed when utilizing TDNIRS37 (see below). The confounding effects of an increased skin blood flow on oxygenation variables [in particular [oxy (Hb+Mb)] determined by CW-NIRS] have been confirmed in a study carried out by Messere and Roatta.70 These authors observed that most of the warming-indud increase of the sum between oxy- and deoxy-Hb and -Mb ([total(Hb+Mb)]) resulted from an increase in [oxy(Hb+Mb)], with a relatively small contribution by [deoxy(Hb+Mb)]. They also observed that the "contamination" of the muscle NIRS signal by an increased skin blood flow was substantially redud by using spatially resolved (SRS) NIRS (see below). No increases in muscle O2 saturation ( , see below) were observed, by utilizing SRS-NIRS, in the vastus lateralis of subjects performing dynamic knee-extension exercise at 20% of MVC after thigh heating at 37°C and 42°C.71 The unchanged was observed in the presen of a marked increase in cutaneous vascular conductan.54 Several studies have confirmed the presen of a good or very good correlation between the [deoxy(Hb+Mb)] signal, or other tissue oxygenation variables determined by NIRS, and venous O2 saturation, both in an animal model of isolated muscle in situ14,72,73 and in exercising humans.20,30,74,75 In Wüst et al.,72 the correlation was worse during the initial part of contraction period, which in that model is associated with a forful "muscle pump" effect and a substantial blood squeezing resulting from the sudden onset of tetanic contractions. The first NIRS instruments widely utilized on skeletal muscle adopted, as a deoxygenation index, the differential absorption of light between 760 and 800 (or 850) nm.52,53,67,76­83 The approach was based on the absorption characteristics of NIR light by the chromophores of interest at the two wavelengths. Subsequent instruments allowed the obtainment of relative (with respect to an initial value arbitrarily set equal to zero) or absolute [oxy(Hb+Mb)] and [deoxy(Hb+Mb)] values. The sum of [oxy(Hb+Mb)] and [deoxy(Hb+Mb)] indicates the total Hb + Mb volume ([total(Hb+Mb)]) in the tissue under consideration. Sin total [Mb] cannot change acutely during exercise, changes in [total(Hb+Mb)] would reflect a vasodilation or an increased capillary hematocrit in the tissue under consideration. Absolute values of the above-mentioned variables can be obtained by the more technologically sophisticated (and more expensive) SRS, TD, or frequency-domain (FD) instruments, whereas the less technologically sophisticated, less expensive, and more widely utilized CW instruments allow only relative values (for a more detai discussion on this topic, see below and Ferrari et al.18,21,24). This represents a limitation, which can be at least mitigated by performing a Journal of Biomedical Optics "physiological calibration" at the end of each test, by a transient ischemia of the investigated limb, obtained by applying for a few minutes a markedly suprasystolic pressure by a cuff, "upstream" of the region of investigation (see, e.g., Fig. 1 in Porlli et al.84). This maneuver is not too uncomfortable for the subject. The range of values of [deoxy(Hb+Mb)] obtained in the muscle between rest and the condition of full extraction (i.e., when the [deoxy(Hb+Mb)] signal reaches a "plateau" after 4 to 5 min of ischemia) should be related to the range of Cða-vÞO2m values between rest and full extraction. By adopting this physiological calibration, "semiquantitative" fractional O2 extraction values can be obtained, also by CW-NIRS instruments. Some controversy has been raised85,86 about which one of the two variables ([oxy(Hb+Mb)] or [deoxy(Hb+Mb)]) should more precisely reflect changes in skeletal muscle fractional O2 extraction. As discussed by Grassi,87 the time-course of [deoxy(Hb +Mb)] (while not that of [oxy(Hb+Mb)]) during a constant work rate exercise65 qualitatively reflects, rather closely, the time courses of variables related to fractional O2 extraction across a wide spectrum of experimental models, such as Cða-vÞO2 determined across a contracting isolated in situ muscle preparation88 or an exercising limb,11,20 microvascular PO2 (PO2mv ) determined by phosphoresn quenching in rat spinotrapezius muscle,89 intrallular PO2 determined by phosphoresn quenching in isolated amphibian muscle fiber.90 Similar kinetics between PO2mv and [deoxy(Hb+Mb)] during electrically stimulated contractions in the rat gastrocnemius were observed.91 A study described a very similar pattern, also, for deoxy-Mb determined by 1 H-MRS17. In other words, during constant work rate exercise (or isometric contractions of constant metabolic rate), a series of variables related to O2 extraction, spanning from Cða-vÞO2 across a whole limb to Mb saturation, show a very similar time-course: unchanged versus rest for a few seconds (suggesting an adequacy of _ _ QO2m with respect to VO2m --this information is relevant in _ terms of the factors determining the VO2 kinetics3,4,10), then _ a rapid exponential increase (the increase VO2m exeds the _ increase in QO2m ) to a new steady state, in which the ratio _ _ VO2m QO2m reaches a new equilibrium, although at a higher value compared to that at rest. In other words, [deoxy(Hb+Mb)] (while not [oxy(Hb+Mb)]) belongs to this family of variables related to fractional O2 extraction. This indirectly but strongly supports the role of [deoxy(Hb+Mb)] as an estimate of skeletal muscle fractional O2 extraction. The conpt is further strengthened by the issue of [oxy(Hb+Mb)] increases deriving from the enhand blood flow to the skin for thermoregulatory purposes, as discussed above. Strictly speaking, an increased [deoxy(Hb+Mb)] can be considered an estimate of an increased fractional O2 extraction only if [total(Hb+Mb)] is constant, which is not always the case in exercising skeletal muscles. However, there is ample eviden14,18,65,86,92­94 suggesting that changes in [deoxy(Hb+ Mb)] are less influend by changes in [total(Hb+Mb)] compared to changes in [oxy(Hb+Mb)]. Adami et al.92 observed a good correlation between the increase in [deoxy(Hb+Mb)] and the decrease in femoral blood flow during passive head_ up tilting, in the presen of a presumably unchanged VO2m . The data confirm the role of changes in [deoxy(Hb+Mb)] in evaluating O2 extraction in the presen of acute and passively _ indud changes in QO2m . In the study, passive head-up tilt indud also an increased venous blood volume, which September 2016 091313-6 Vol. 21(9) Grassi and Quaresima: Near-infrared spectroscopy and skeletal muscle oxidative function. . . complicated the interpretation of [deoxy(Hb+Mb)] as an index of O2 extraction;92 venous blood pooling is, however, unlikely to occur during exercise. In any case, the issue of the influen of blood volume has been substantially overcome by a rent study94 in which the authors calculated, on the basis of the assumption that during an arterial occlusion changes in [oxy (Hb+Mb)] and [deoxy(Hb+Mb)] should occur with a 11 ratio, a useful "blood volume correction factor," which can be applied to both [oxy(Hb+Mb)] and [deoxy(Hb+Mb)]. As mentioned above, SRS-, TD-, or FD-NIRS instruments allow the measurement of absolute values of [oxy(Hb+Mb)] and [deoxy(Hb+Mb)].24 By utilizing these values, a tissue oxygenation index (TOI) can be calculated, as the ratio [oxy (Hb+Mb)]/[total(Hb+Mb)].95 As an oxygenation variable, TOI is appealing sin it is expressed as a perntage, similarly to arterial O2 saturation (SO2a ). Sin it includes [oxy(Hb+ Mb)], however, also TOI may be more significantly influend by skin blood flow compared to [deoxy(Hb+Mb)]. Vogiatzis et al.30 utilized it to evaluate the intramuscular match_ _ ing between VO2m and QO2m . 3.4.2 Skeletal muscle O 2 consumption Physiological Variables of Functional Evaluation Which of the variables in the Fick equation [Eq. (1)] can be at least estimated by NIRS? The answer is: all of them, although in _ _ order to determine VO2m or Qm some specific (and rather unphysiological) or invasive protocols need to be adopted. When these protocols are not utilized, the oxygenation indis _ obtained by NIRS evaluate the dynamic balan between VO2m _ and QO2m . In other words, these indis allow the estimation of skeletal muscle fractional O2 extraction, a proxy of Cða-vÞO2m (see also below). 3.4.1 Skeletal muscle (or regional) blood flow De Blasi et al.96 determined forearm Q by NIRS, at rest and after hand exercise, by inducing a 50-mmHg venous occlusion by a cuff. They reasoned that if venous outflow from a muscle is impeded by the cuff, whereas arterial inflow is not (or at least is only partially impeded), the increase in [total(Hb _ +Mb)] as a function of time should be related to Q. The authors _ observed indeed an exllent correlation between forearm Q, determined by NIRS as described above, and the increase in forearm volume determined by plethysmography. A similar approach was adopted by van Beekvelt et al.32 No comparison _ with other methods to determine Qm was made. Limitations of the approach relate to the fact that pressure by the cuff inevitably limits, although only in part, also arterial inflow, as rently _ confirmed.97 Moreover, regional Q measurements could be carried out only during the recovery from exercise, and it is well known that the variable may rapidly change in the recovery phase.6 _ Qm in the exercising calf muscles has been determined in humans by utilizing NIRS in association with the intravenous infusion of the NIR trar indocyanine green (ICG).98 ICG is a fluoresnt dye approved by the U.S. Food and Drug Administration and European Medicines Agency for human use. Arterial blood was withdrawn by a pump and [ICG] was detected by photodensitometry, whereas NIRS optodes positioned over the muscle detected ICG transit in the microcircu_ lation at several wavelengths. Qm measurements were compared with those obtained by established methods. The main drawback of this elegant approach resides in its invasiveness, related to the need of obtaining arterial blood samples. The method has been _ utilized to determine Qm in respiratory99 and skeletal muscles100. Journal of Biomedical Optics Hamaoka et al.101 determined by NIRS the initial rate of Hb and Mb deoxygenation in finger flexors of humans during ischemia performed at rest and immediately after submaximal exercise of different intensities, and considered this variable a reflection of _ VO2m . These authors observed a significant correlation between this rate and the conntrations of intramuscular metabolites (PCr and adenosine diphosphate), determined by 31 P-MRS, considered to be regulators of oxidative phosphorylation. They concluded that NIRS can quantify the rate of oxidative metabolism at rest and after exercise. The validity of the approach was subsequently confirmed.102 The limitations are represented by the fact that the NIRS measurements need to be carried out in ischemic conditions and that the measurements are possible only in _ the recovery phase following exercise, during which VO2 rap6 idly decreases. While ischemic contractions do not represent a physiological condition, a "less unphysiological" experimental model would be one in which blood flow is held constant. Also in this case, according to the rearranged Fick equation [Eq. (2)], and after considering [deoxy(Hb+Mb)] a proxy of Cða-vÞO2m , the increase in [deoxy(Hb+Mb)] should represent an estimate of _ VO2m . This has indeed been experimentally confirmed in the study by Wüst et al.,72 which carried out in an isolated dog gas_ trocnemius in situ preparation. In the study, Qm was held con_ stant by a pump, VO2m was calculated by the Fick equation across the muscle and NIRS optodes were directly applied on the surfa of the exposed muscle. During metabolic transitions, the kinetics of adjustment of [deoxy(Hb+Mb)] was very similar _ to the kinetics of adjustment of VO2m . Brief sequential ischemic periods carried out during the recovery from exercise/contractions have been utilized in order to determine, by NIRS, the kinetics of recovery of _ VO2m following exercise.94,103­106 Also in this case, the rate of increase in [deoxy(Hb+Mb)] (and/or the rate of decrease in [oxy(Hb+Mb)]) during each of the brief ischemic periods _ is considered an estimate of VO2m . The kinetics of recovery _ of VO2m , as determined by the abovementioned protocol, has been shown to be closely correlated with two well-established variables of functional evaluation of skeletal muscle oxidative metabolism (frequently and incorrectly termed "oxidative capacity" by some of these authors): (1) the kinetics of recovery of [PCr], as determined by 31 P-MRS following exercise104 and (2) maximal adenosine diphosphate-stimulated mitochondrial respiration, as determined by high-resolution respirometry in isolated and permeabilized skeletal muscle fibers.106 3.4.3 Skeletal muscle fractional O 2 extraction As discussed above, in "normal" experimental conditions, oxygenation variables obtained by NIRS allow the evaluation of the _ _ dynamic balan between VO2m and QO2m , in other words of skeletal muscle fractional O2 extraction, a proxy of Cða-vÞO2m . NIRS oxygenation variables can represent only a proxy of Cða-vÞO2m sin they are usually expressed in relative terms, and the proportional contributions of arterial and venous blood to the overall signal are unknown. In the next paragraphs, we will provide examples of conditions in which analysis of September 2016 091313-7 Vol. 21(9) Grassi and Quaresima: Near-infrared spectroscopy and skeletal muscle oxidative function. . . skeletal muscle fractional O2 extraction appears relevant and of physiological interest. The capacity for prolonged exercise depends upon the capacity to supply the exercising muscles with O2 in order to satisfy the metabolic requirements. During exercise, however, fractional O2 extraction increases. In other words, compared to rest, the muscles (as other organs) utilize a greater perntage of the O2 they reive by the cardiovascular system. We will discuss, later in this section, how this increase in fractional O2 extraction could impair peripheral O2 diffusion and oxidative metabolism through the resulting decrease in microvascular PO2 (PO2mv ). Textbook physiology says that at the systemic level, in humans at rest, fractional O2 extraction is 25%, whereas during maximal exercise, the variable increases to 75%. In steady_ state conditions during exercise, the relationship between Q and _ _ VO2p is linear, with a slope of 5 (1 L min-1 increase in VO2p is _ accompanied by a 5 L min-1 increase in Q). Considering that 15 of the volume of arterial blood is represented by O2 (CaO2 200 mL L-1 of blood), the linear relationship between _ _ QO2 and VO2p has a slope of 1. As a consequen of the linear _ _ _ Q (or QO2 ) versus VO2p relationship, systemic Cða-vÞO2 would increase hyperbolically as a function of work rate. This has indeed been observed during ramp incremental cycling exercise.107 _ _ The relationship between Qm O2 and VO2m is somehow different when determined at the microvascular level, which is the domain investigated by NIRS. During an incremental or ramp exercise, the NIRS-derived [deoxy(Hb+Mb)] follows a sigmoid pattern,62,84,107­113 with a shallow increase at low work rates (sug_ _ gesting a tighter matching between muscle VO2m and QO2m , presumably associated with the recruitment of more oxidative fibers), a linear intermediate portion (suggesting greater O2 _ _ extraction and a progressively higher VO2m QO2m, presumably associated with the recruitment of less oxidative fibers), followed _ by a plateau at work rates approaching 80% of VO2m;max . At submaximal work rates, before the "plateau" is reached, the observation of higher fractional O2 extraction for the same _ VO2m (or work rate), in other words, a left-shifted [deoxy(Hb _ +Mb)] versus work rate curve would suggest an impaired QO2m . On the other hand, a lower fractional O2 extraction (right-shifted [deoxy(Hb+Mb)] versus work rate) would suggest an exss _ _ QO2m with respect to VO2m . This information may have profound physiological and pathophysiological significan (see below). The sigmoid shape of the [deoxy(Hb+Mb)] versus work rate curve has been shown to vary in muscles characterized by different fiber type compositions49 and has been confirmed (with minor differens) also during supine exercise114 as well as during incremental exercise in acute hypoxia.115 A right-shifted [deoxy(Hb+Mb)] versus work rate curve was described in trained cyclists.116 The [deoxy(Hb+Mb)] kinetics during constant work rate exercise has been utilized to evaluate the effects on the rate of adjustment of oxidative metabolism by a "prior" or "warm up" or "increased baseline" exercise,72,117­119 or to investigate the effects on microvascular fractional O2 extraction by interventions such as dietary nitrate supplementation, aimed at increasing microvascular nitric oxide (NO) availability.120 For example, the increased muscle oxygenation observed by Gurd et al.118 following a prior heavy intensity exercise suggests an enhand O2 availability, which should have contributed to _ the observed faster pulmonary VO2 kinetics. In other studies, the results were not conclusive. Bailey et al.,117 for example, Journal of Biomedical Optics observed after oral nitrate supplementation, a decreased muscle O2 extraction, which, however, could not be simply attributed to enhand O2 delivery sin it was associated with a decreased _ VO2 . Skeletal muscle fractional O2 extraction determined by NIRS can also give valuable physiological and pathophysiological insights into the integrated responses to exercise (and to other conditions) involving different muscle groups or skeletal muscles and other organs, as well as into the physiological _ mechanisms of Qm regulation. _ For example, arm Q (by thermodilution) and muscle oxygenation (by NIRS) were measured at the transition from _ arm to arm + leg exercise.66 During maximal exercise, arm Q _ decreased at the transition, to a degree that VO2m;max and muscle oxygenation were compromised. Thus, NIRS can give valuable _ _ insights into the coordination of Qm and VO2m adjustments to exercise between different muscle groups, in conditions of _ increased O2 demands and/or limited QO2m (see also below). Turner et al.121 observed [deoxy(Hb+Mb)] increases both in limb locomotor muscles and in respiratory muscles after interventions, such as inspiratory muscle loading performed during _ cycling exercise at 80% of VO2p;max , or after increasing work _ rate from 80% to 100% of VO2p;max . The results suggest a lim_ ited QO2m , both in locomotor and in respiratory muscles, which may impair exercise toleran during maximal exercise or in conditions of increased respiratory muscles work. By applying lower body negative pressure, Soller et al.122 indud a progressive decrease of ntral blood volume and a decreased stroke volume, which were paralle by a decreased muscle oxygenation from vasoconstriction. According to these authors, skeletal muscle deoxygenation determined by NIRS could be utilized as an early indicator of ntral hypovolemia and impending cardiovascular collapse. NIRS has also been utilized to elucidate basic physiological _ mechanisms of Qm regulation, such as the metabolic modulation of sympathetic vasoconstriction (functional sympatholysis) by exercise123 or by tissue hypoxia.124 By simultaneously measuring sympathetic nerve activity by microelectrodes and forearm oxygenation by NIRS, Hansen et al.123 provided eviden in favor of functional sympatholysis. Reflex sympathetic activation, which consistently decreased oxygenation in resting forearm muscle, had no effect on oxygenation when the muscles were exercised. The [deoxy(Hb+Mb)] plateau during an incremental exercise can be considered an estimate of the maximal capacity of O2 extraction during exercise and has been utilized in several studies84,109­113,125­127 as a variable of functional evaluation of skeletal muscle oxidative metabolism. Venous blood draining from exercising muscles still contains small but significant amounts of O2 ; in other words, fractional O2 extraction is not 100%.128­130 Which are the normal values of fractional O2 extraction across skeletal muscle during maximal exercise? According to Knight et al.,128 during cycling, the value can go up to 90%, as determined by invasive measurements carried out across an exercising limb. A fractional O2 extraction value of 85%, also in this case determined invasively across an exercising limb, was observed during maximal knee-extensor exercise,130 which is an exercise paradigm (involvement of a relatively low small muscle mass) in which cardiovascular constraints to O2 delivery are not present or are significantly redud, and skeletal muscle blood flow can be three times higher than during conventional cycle ergometer exercise. September 2016 091313-8 Vol. 21(9) Grassi and Quaresima: Near-infrared spectroscopy and skeletal muscle oxidative function. . . These values are very similar to those obtained in normal subjects by NIRS for [deoxy(Hb+Mb)] at peak exercise, expressed as a perntage of the maximal [deoxy(Hb+Mb)] value observed during a transient limb ischemia.84 _ Although, in normal subjects, VO2p;max is traditionally con_ sidered to be mainly limited by maximal cardiac output (Qmax ) _ and maximal cardiovascular O2 delivery (QO2 max ),131 the role of peripheral factors cannot be overlooked.132,133 If one considers the Fick equation [see Eq. (1)] applied to conditions of maximal exercise, it is obvious that the main differen between subjects _ _ with high VO2p;max and subjects with low VO2p;max resides in _ 2 max , and not in Cða-vÞO2 max . However, the conpt that QO _ QO2 max represents cardiovascular limitations and Cða-vÞO2 max or maximal fractional O2 extraction represent peripheral factors is an oversimplification.134 In an in-series O2 transport system, every step must play a role in affecting the overall outcome, and _ the different functions are strictly interconnected.133 QO2 , by setting microvascular PO2 (PO2mv ), determines peripheral O2 diffusion and therefore fractional O2 extraction. In the presen _ of an elevated QO2 , as it occurs in athletes or after training, peripheral O2 diffusing capacity (DO2m ) must increase in order to allow a large fractional O2 extraction. Although the issue is debated,135 in skeletal muscle, an O2 _ extraction limit is present, which contributes to VO2p;max and _ VO2m;max limitation; moreover, a limitation in peripheral O2 diffusion appears to be the major factor responsible for the limited extraction.133 Exercise training may increase muscle fractional O2 extraction by elevating muscle(s) DO2m to a greater extent _ than QO2m;max .35,42 Kalliokowski et al.136 observed by positron emission tomography that fractional O2 extraction during exercise is higher in enduran trained athletes compared to untrained controls. According to Whipp and Ward,137 systemic _ Cða-vÞO2 max increases hyperbolically with VO2p;max . In other words, also in healthy subjects, fractional O2 extraction is rel_ evant in determining VO2p;max . This conpt applies even more strongly to patients or to conditions in which oxidative metabolism is impaired (see below). The "snario" depicted above can be looked at also from another perspective: an increased O2 extraction may impair peripheral O2 diffusion. This could occur also during submaximal exercise. A fall in oxygenation (corresponding to an _ _ increased VO2m QO2m ) suggests a less pronound or inad_ equate vascular response in relation to an increased VO2m . This would determine a decreased PO2mv and mean capillary PO2 , a decreased driving pressure for peripheral O2 diffusion from capillaries to mitochondria,42,43,138 and a functional impairment of skeletal muscle oxidative metabolism. The phenomenon could also occur transiently in the early phase of a constant work rate exercise. This is suggested by the transient "overshoot" of skeletal muscle fractional O2 extraction (usually determined by analyzing the [deoxy(Hb+Mb)] time-course), which has been described in contracting in situ dog muscle14 as well as during constant work rate exercise in different populations (see also below), such as patients with chronic heart failure (CHF),95,139 patients with metabolic myopathies,140 type 2 diabetes,141 chronic obstructive pulmonary disease,142 healthy subjects undergoing bed rest deconditioning84 or bed rest associated with hypoxia,127 healthy subjects during arm exercise.81 In the latter study, the overshoot in skeletal muscle fractional O2 extraction, determined by NIRS, was closely associated with an overshoot in Cða-vÞO2 , determined invasively across the exercising muscles. Goodwin Journal of Biomedical Optics _ et al.14 experimentally slowed QO2m kinetics in isolated in situ dog muscle and observed an enhand [deoxy(Hb+Mb)] over_ shoot and a slowed VO2m kinetics. In human subjects acutely exposed: to normobaric hypoxia, Bowen et al.33 observed a slower VO2p kinetics and a more pronound [deoxy(Hb+Mb)] transitory increase, although the magnitude of the overshoot was unaffected. The O2 extraction overshoot, occurring early during a constant work rate exercise, is considered to be caused by a _ _ transient mismatch between QO2m and VO2m .42,43,138,143,144 The mismatch could derive from a suboptimal NO signaling within the muscle, which could "uncouple," both temporally and spa_ tially, the heterogeneous microvascular QO2m increase from the _ 2m increase.43,138 presumably heterogeneous VO The transiently increased fractional O2 extraction is associated with a transiently decreased PO2mv .42,43,138,144 This phenomenon has been described in animal models, in which PO2mv was determined by phosphoresn quenching.35,145 As mentioned above, the decreased PO2mv would redu the driving pressure for peripheral O2 diffusion and impair oxidative metabolism. The impairment would be functionally significant also because it would be temporally located in the critical early phase following an increase in metabolic intensity.3,4,6,10 A partially different approach to the [deoxy(Hb+Mb)] overshoot has been taken by Murias et al.146 These authors utilized _ a "normalized" ½deoxyðHb þ MbÞVO2p ratio, in which both variables are expressed as a perntage of the response. In _ order to calculate the ratio, the VO2p signal is left-shifted by 20 s, which is assumed to represent, on average, the delay _ between the VO2m increase (the variable of interest in the com_ parison with [deoxy(Hb+Mb)]) and the VO2p increase,3,4 which is the variable usually determined. Another assumption related _ _ to this approach is that VO2p kinetics equals VO2m kinetics. This is not exactly true (see the discussion in Koga et al.42): the two variables have similar kinetics11 but with a substantial intersub_ ject variability. Moreover, [deoxy(Hb+Mb)] and VO2p increases are, in relative terms, substantially different, and therefore, the normalization (perntages of responses) likely introdus a distortion in the calculation of the ratio. In another approach, Ferreira et al.143 assumed (as Murias _ _ et al.146 did) that VO2p kinetics equals VO2m kinetics, and that [deoxy(Hb+Mb)], as an estimate of O2 extraction, quantitatively reflects Cða-vÞO2 ; the authors then calculated microvascular blood flow adjustment by solving the Fick equation. The main limitations of this approach, in our opinion, reside in the _ _ assumptions: the first one (VO2m kinetics equals to VO2p kinetics) has been discussed above, but also the second one ([deoxy(Hb+Mb)] as a quantitative measurement of Cða-vÞO2 ) is not strictly correct: even when convincing "physiological calibration" methods are utilized, [deoxy(Hb+Mb)] allows at best only a semiquantitative estimation of Cða-vÞO2 , and therefore, its use in replacing Cða-vÞO2 in mathematical equations seems questionable.147 Moreover, the approach by Ferreira et al.143 assumes that the contribution by Mb to the NIRS signals is zero; as discussed above, this is likely not the case. _ During the recovery following exercise, the decrease in VO2p _ and VO2m follows an exponential time course, even in the presen of a sudden decrease in mechanical power output.6 _ In these conditions, the "exss VO2m ," with respect to the needs of myosin ATPase, derives essentially from the resynthesis of PCr, which occurs by oxidative phosphorylation. Upon the assumption of equilibrium of the CK reaction, the recovery rate of [PCr] following exercise is therefore a function of September 2016 091313-9 Vol. 21(9) Grassi and Quaresima: Near-infrared spectroscopy and skeletal muscle oxidative function. . . mitochondrial ATP production. The rate of recovery of [PCr] following exercise has often been utilized as a tool of evaluation of skeletal muscle oxidative metabolism.15,104 Another variable frequently utilized as a functional evaluation tool is the NIRSdetermined reoxygenation kinetics during the recovery. This kinetics is a function of the interplay between the kinetics of _ _ QO2m and the kinetics of VO2m , and cannot discriminate between the two. However, a good agreement was found between the rate of recovery of [PCr] (as determined by 31 P-MRS) and the reoxygenation kinetics (as determined by NIRS) following moderate-intensity exercise that is the absen of significant drops in pH.80 These authors concluded that the reoxygenation kinetics during the recovery from a moderateintensity exercise could represent a functional evaluation tool of oxidative performan. 3.4.4 Other variables and purposes Several studies have been conducted with the aim to identify, by NIRS, variables of functional evaluation of oxidative metabolism (thresholds), such as the ventilatory threshold,148,149 the "EMG threshold,"150 the onset of blood lactate accumulation during incremental exercise,151 or the "maximal lactate steady state."152 Most of these studies observed a ni correlation between NIRS-obtained indis and more traditional variables utilized to identify the various thresholds.148,149,151,152 Broxterman et al.153 utilized NIRS, together with EMG and the analysis of the curvature constant of the power versus time to exhaustion relationship, in order to evaluate the role of ntral and peripheral fatigue during exercise with small muscle mass (handgrip) in conditions of blood flow occlusion. ntral (brain) factors were identified as responsible for fatigue during submaximal elbow flexors exercise in severe hypoxia;154 in the study, the authors utilized muscle and brain NIRS, evoked and EMG responses to electrical muscle stimulation, transcranial magnetic stimulation, and motor-evoked potentials. Soller et al.122 developed a mathematical model, which correlated NIRS spectra with muscle pH (pHm ) values obtained invasively by sensors plad in the muscle. They could then predict pHm values on the basis of NIRS spectra, and determined, during incremental exercise, an "Hþ threshold," which correlated with classic "metabolic thresholds". Towse et al.155 utilized NIRS blood volume and muscle oxygenation changes to confirm the blood-oxygenation-leveldependent (BOLD) mechanism underlying the changes of magnetic resonan imaging signal intensity observed following brief, single contractions. Several NIRS studies have been performed in electrically stimulated contractions. For instan, McNeil et al.156 observed by NIRS a greater metabolic demand, associated with greater fatigue during electrically stimulated (versus voluntary), contractions carried out at the same torque production. On the contrary, the local O2 demand of the bips brachii was found similar between electrical muscle stimulation and maximal voluntary isometric contractions.157 versus young activity-matched controls, in association with _ _ higher Qm and QO2m and an unchanged microvascular fractional O2 extraction. These results point to a higher energy cost of contractions as the main factor responsible for the redud exercise capacity in the elderly. At least in part, different results were described by Ferri et al.,125 who observed in 78-year old subjects, higher skeletal muscle (vastus lateralis) fractional O2 extraction, at the same absolute work rate, suggesting an _ impairment of QO2m . This phenomenon was observed both during cycle ergometer exercise (in which the limitations to oxidative metabolism are mainly related to cardiovascular O2 delivery) and during one-leg knee-extension exercise,125 in which cardiovascular limitations are substantially redud.130 _ VO2p kinetics is slower in the elderly. Murias et al.159 observed that this slower kinetics was associated with a larger _ overshoot in the ½deoxyðHb þ MbÞVO2p (both variables were expressed as a perntage of their total response, see also above) ratio, suggesting, according to the authors, an impaired adjust_ ment of QO2m . This would be in agreement with observations of a blunted vasodilatory dynamics in arterioles of old rats.160 _ Exercise training speeded VO2p kinetics and redud the _ 2p overshoot,159 suggesting an improved ½deoxyðHb þ MbÞVO _ _ matching between QO2m and VO2m . In another study, no _ ½deoxyðHb þ MbÞVO2p overshoot was observed in chronically trained elderly subjects.161 It should be considered, however, that in these studies,159,161 the overshoot in [deoxy(Hb _ +Mb)] was only relative to VO2 ; in other words, no "real" [deoxy(Hb+Mb)] overshoot was present. Immobilization/Microgravity and Countermeasures Higher skeletal muscle fractional O2 extraction for the same work rate, a redud peak capacity of O2 extraction during an incremental exercise, and an overshoot of [deoxy(Hb+Mb)] during constant work rate exercise were observed in healthy young subjects undergoing 35 days of bed rest.84,111 These findings were observed during both cycle ergometer exercise84 and one-leg knee extension exercise.111 These results include skeletal muscle oxidative metabolism among the "victims" of profound inactivity. Sin bed rest studies have been considered a "proxy" of microgravity (see the discussion in Porlli et al.,84 Salvadego et al.111), the same conpt applies to microgravity. Superposition of normobaric hypoxia (equivalent to an altitude of about 4000 m) upon 10 days of bed rest attenuated, during one-leg knee extension exercise (but not during cycle ergometer exercise), the impairment of the peak capacity of fractional O2 extraction and the magnitude of the overshoot, which were observed following bed rest alone.127 An enhand perfusive and diffusive O2 delivery deriving from the hypoxia-indud increased [Hb] may explain, at least in part, this unexpected finding. Training programs carried out during 21-day forearm immobilization by a plaster cast were found effective in preventing the decline in muscle oxidative function, enduran, and strength.162 Aging, Immobilization, Hypoxia Aging Hypoxia By combining 31 P-MRS, Doppler ultrasound imaging, and NIRS Layec et al.158 observed a higher ATP cost of contractions during plantar flexion exercise in elderly subjects (73-year old) Journal of Biomedical Optics During acute moderate normobaric hypoxia (inspired O2 partial pressure [PO2I ] 118 mmHg, equivalent to an altitude of 2500 m), muscle deoxygenation during an incremental cycle exercise is not significantly different, for the same absolute work rate, compared to normoxic conditions.163 The data September 2016 091313-10 Vol. 21(9) Grassi and Quaresima: Near-infrared spectroscopy and skeletal muscle oxidative function. . . _ suggest higher Qm in hypoxia compared to normoxia and, thus, the existen of control mechanisms, which effectively match _ _ QO2m to VO2 . This effective matching is presumably lost at higher altitudes. Billaut et al.164 collected muscle and brain oxygenation data to gain insights into the ntral and peripheral factors affecting muscle fatigue in conditions of limited O2 delivery. They described a trend toward lower vastus lateralis muscle oxygenation in subjects performing repeated sprints and acutely exposed to a slightly more pronound (compared to that of the previously mentioned study) moderate hypoxia (FO2I ¼ 0.138, PO2I 98.4 mmHg). Subudhi et al.165 described greater fractional O2 extraction values for the same absolute work rate in subjects acutely exposed to hypobaric hypoxia (PO2I 86 mmHg) corresponding to an altitude of 4300 m. Interestingly, no differens versus acute hypoxia were described, in the same subjects, in chronic hypoxia (5-day acclimatization at 1830 m and 1-day at 4300 m). In all the mentioned studies,163­165 marked differens between normoxia and acute or chronic hypoxia were described for prefrontal cortex oxygenation. Unchanged muscle oxygenation during exercise was described after versus before a 72-day acclimatization to chronic hypobaric hypoxia (Mt. Everest expedition);166 the unchanged muscle deoxygenation was associated with an increased hypoxic toleran and with enhand acute hypoxic ventilatory response and rebral oxygenation. In another Mt. Everest expedition, a slower muscle (thenar eminen) reoxygenation kinetics was observed during vascular occlusion tests carried out at 4900 and 5600 m.167 A more pronound muscle deoxygenation during constant work rate exercise was described during acute hypoxia,67 obtained either by having the subjects breath air with a FO2I of 0.15 or by carbon monoxide loading aimed at increasing carboxyhemoglobin saturation. Interestingly, the aclerated deoxygenation was described only during exercise above the "lactate threshold," that is, at an exercise intensity at which O2 supply to the exercising muscles becomes critical.2,3,4,10 A greater deoxygenation of acssory respiratory muscles during voluntary isocapnic hyperpnea was observed in hypoxia (FO2I ¼ 0.10 - 0.11) versus normoxia,168 in association with a higher muscle activation determined by EMG. Patients Populations In this section, we will present an overview of some representative studies in which the variables of functional evaluation discussed in the previous section have been determined in adult patients' populations. The aims were to evaluate the evolution of impairments as a function of time, to identify correlations with clinical symptoms, to evaluate the effects of therapeutic or rehabilitative interventions, and to gain pathophysiological and diagnostic insights. A systematic review on muscle NIRS measurements in the clinical care of term and preterm neonates has been rently published.169 Chronic Heart Failure Several studies have been conducted by utilizing NIRS in patients with CHF. The complex interplay between ntral and peripheral factors in determining the pathophysiology of the disease and the impaired exercise toleran in CHF patients has been discussed in detail in the review by Poole et al.138 A subsequent review by the same group144 discussed, from Journal of Biomedical Optics the same perspective, the effects of training in this patient population. Wilson et al.62 observed higher fractional O2 extraction, for the same absolute work rate, in CHF patients versus agematched healthy controls; the results suggest an impaired _ QO2m in the patients, presumably a consequen of the impaired cardiac function. A similar pattern was observed by another study in CHF patients.126 Interestingly, in this study, the _ impaired QO2m response was not affected by 3 months of light-to-moderate exercise training. The training intervention, on the other hand, increased the maximal capacity of skeletal muscle fractional O2 extraction during exercise, allowing the patients to reach values not different from those of controls. A very similar pattern to that described in CHF patients126 before training was observed in patients evaluated after a sucssful heart transplantation,109 carried out in the treatment of severe CHF. The data suggest that the surgical intervention, although improving cardiac function, did not significantly _ improve the QO2m impairment at submaximal exercise and the redud capacity of maximal fractional O2 extraction by skeletal muscles. Also in patients undergoing heart transplantation, a complex interaction between ntral and peripheral functions limits exercise toleran.109 Sperandio et al.139 described by NIRS in CHF patients, during constant work rate submaximal exercise, a transient overshoot in fractional O2 extraction (increased [deoxy(Hb+Mb)]), presumably leading to a decreased PO2mv and to an impaired peripheral O2 diffusion during the critical early phase of the metabolic transition. This overshoot could derive from a suboptimal intramuscular NO signaling.138 The [deoxy(Hb+Mb)] overshoot is qualitatively equivalent and has the same pathophysiological meaning with respect to the transient PO2mv undershoot described in the spinotrapezius muscle of CHF rats.170 In addition, Sperandio et al.139 observed that in CHF patients an increased NO bioavailability, obtained by the administration of sildenafil, eliminated the [deoxy(Hb+Mb)] overshoot, determined a faster _ VO2p kinetics, and improved exercise toleran. Bowen et al.95 observed that the deoxygenation overshoot in CHF patients was attenuated by a prior exercise (aimed at enhancing _ _ QO2m ), which determined also a faster VO2p kinetics, confirming the link between the overshoot and an impaired oxidative metabo_ lism. On the basis of the deoxygenation and VO2p kinetics, the authors identified two subgroups of patients: the most severely limited ones were those with an impaired skeletal muscle oxidative metabolism.95 Belardinelli et al.82 observed in CHF patients a slower reoxygenation kinetics following exercise, in association with a _ slower VO2p kinetics. In another study, Hanada et al.171 observed that the slower reoxygenation kinetics in CHF patients was associated with a markedly slower kinetics of recovery of [PCr], as determined by 31 P-MRS. As mentioned above, a slower reoxygenation kinetics during the recovery after exercise is considered an index of impaired skeletal muscle oxidative performan.80 At least in part, different results were obtained by Kemp et al.:172 according to these authors, in CHF patients the slowing of muscle reoxygenation kinetics was more pronound than the slowing of [PCr] recovery; the data suggest _ a QO2m limitation. _ A slower recovery kinetics of VO2m (determined by NIRS by utilizing the method by Ryan et al.94 mentioned above) following wrist-flexor exercise was observed in systolic CHF patients;173 interestingly, whereas in control subjects, a 4September 2016 091313-11 Vol. 21(9) Grassi and Quaresima: Near-infrared spectroscopy and skeletal muscle oxidative function. . . week period of exercise training determined a faster recovery kinetics, this did not occur in the CHF patients. The authors concluded that the impairment of nonlocomotor muscles oxidative metabolism in the CHF patients was not reversible with a short period of exercise training. Moreover, the impairment was not simply due to deconditioning, but suggests a skeletal muscle dysfunction intrinsically associated with the disease, as also proposed in the reviews by Poole et al.138 and Hirai et al.144 Mancini et al.76 observed deoxygenation of acssory muscles of respiration (serratus anterior) in CHF patients (but not in controls) during submaximal and maximal cycle exercise; the observed deoxygenation may contribute to the exertional dyspnea typically described in these patients. It is well known that more severely affected CHF patients typically have a hyperventilatory response to exercise, which holds clinical and prognostic implications.174 The hyperventilatory response, the decreased respiratory muscle strength, and the _ increased work of breathing would increase QO2m requirements of respiratory muscles, and could indu a "competition" for the _ limited available QO2 between respiratory and locomotor muscles, contributing to the decreased exercise toleran.175 In support of this hypothesis, Borghi-Silva et al.176 observed that respiratory muscles unloading, obtained in CHF patients by proportional assisted ventilation, increased exercise toleran in association with an enhand leg muscle oxygenation and blood volume determined by NIRS. These studies demonstrate how NIRS can give valuable pathophysiological insights into the interplay between different muscles in conditions of limited _ available QO2 . A functional evaluation of skeletal muscle oxidative metabolism would be even more important in CHF patients with preserved ejection fraction, in whom peripheral impairments appear more significant than those usually observed in the more "traditional" CHF patients with redud ejection fraction.144,177 In this respect, very few data are available yet. Peripheral Arterial Disease and Diabetes During incremental exercise, PAD patients show near-maximal deoxygenation (with respect to the ischemic calibration) beginning from very low work rates.79 In the same study, the reoxygenation kinetics following exercise was markedly slower in PAD patients; moreover, the half-time of this recovery was significantly correlated with the "ankle-arm index" (ratio of systolic blood pressures at the ankle and at the arm),79 a clinical variable evaluating the severity of the disease. A markedly slower reoxygenation kinetics in PAD patients was confirmed in subsequent studies.83,141 In PAD patients, the involvement of the two legs is often markedly asymmetrical. Interestingly, McCully et al.79 observed that the reoxygenation kinetics was markedly slower in the "bad" leg versus the "good" leg. According to McCully et al.,83 analysis of reoxygenation kinetics by NIRS could be utilized as a screening tool for the presen of PAD. In patients with PAD, NIRS has also been utilized to evaluate the effects of various therapeutic interventions. Whereas Beckitt et al.178 described only modest improvements of muscle oxygenation during exercise in PAD patients treated with angioplasty, substantial positive effects were observed after the administration of sildenafil179 or after an oral supplementation (beetroot jui) of nitrates.180 Both these approaches aimed, although by exploiting different pathways, at increasing NO bioavailability in the microvasculature, thereby taking advantage of Journal of Biomedical Optics the vasodilatory effect of NO and also at improving the intra_ _ muscular matching between VO2m and QO2m . What is the pathophysiological meaning of the slower reoxygenation kinetics in PAD patients? As a general rule, this kinetics is a function of the interplay between the kinetics of _ _ QO2m and the kinetics of VO2m during the recovery following exercise. In PAD patients, the first variable is obviously impaired, but the second one could be impaired as well. This has been demonstrated by Bauer et al.,141 who observed in PVD patients a slower recovery of reoxygenation also in the patients with reasonably normal calf blood flow responses, as evaluated by plethysmography. The clinical relevan of NIRS applications in PAD has been very rently reviewed by Boezeman et al.,181 who considered 183 manuscripts. These authors found sufficient eviden to use NIRS in the clinical setting for assessment of chronic compartment syndrome of lower extremities, and as a surveillan tool for the detection of "free flap failure". On the other hand, the clinical relevan of routine use of NIRS in other vascular diseases was found to be less clear. _ For the same absolute work rate and VO2p , type 1 diabetic patients show (versus controls) higher [deoxy(Hb+Mb)], sug_ _ gesting an impaired QO2m , as also suggested by the lower Qm.182 An overshoot of [deoxy(Hb+Mb)] during constant work rate exercise has been described in patients with type 2 diabetes.183 _ This observation was associated with slower VO2p kinetics and _ appears compatible with an impaired QO2m adjustment, consequen of the microvascular impairment. The [deoxy(Hb+Mb)] overshoot described in diabetic patients is qualitatively equivalent and has the same pathophysiological meaning of the transient PO2mv undershoot described in the exercising muscles of diabetic rats.89 In a review, Pedersen et al.184 discussed the main methods to evaluate mitochondrial dysfunction in patients with PAD and type 2 diabetes. According to these authors, NIRS works better than 31 P-MRS in determining the severity of the impairment. Manfredini et al.185 utilized NIRS in order to find objective correlates between the onset of symptoms of claudication and the limited exercise toleran in PAD patients, with and without diabetes. In order to do so, they determined the area under the [oxy(Hb+Mb)] or [deoxy(Hb+Mb)] curves (with respect to the baseline) during an incremental exercise. These areas evaluate (although in arbitrary units) the overall burden represented by _ _ the dynamic unbalan between VO2m and QO2m . The areas were greater in the PAD patients with diabetes and were correlated with the onset of symptoms. In the PAD patients with diabetes, however, ischemic symptoms were delayed and occurred at a level of deoxygenation which was significantly greater than in the patients with PAD alone, possibly as a consequen of a blunted pain perption and/or of a subjective higher toleran for muscle discomfort associated with diabetes. Chronic Obstructive Pulmonary Disease By substituting nitrogen with helium in inspired air, gas density is redud to 13 of that of normal ambient air. After relieving the work of breathing by having COPD patients breathe a normoxic helium-O2 mixture, Chiappa et al.186 observed less leg _ deoxygenation by NIRS, faster VO2p kinetics, and an increased exercise toleran. The authors hypothesize that by decreasing _ the competition for the available QO2 between respiratory and locomotor muscles, helium-O2 breathing could increase leg _ QO2m , thereby enhancing skeletal muscle oxidative metabolism. September 2016 091313-12 Vol. 21(9) Grassi and Quaresima: Near-infrared spectroscopy and skeletal muscle oxidative function. . . This hypothesis was only in part confirmed in a subsequent study.48 These authors determined by NIRS, by utilizing the ICG dye dilution method,98 intercostal, and quadrips muscles _ Qm in COPD patients breathing room air or a normoxic helium_ O2 mixture. During cycling exercise at 75% of VO2p;max , _ helium-O2 was associated with increases in Qm in both intercostal and quadrips muscles, together with an increased CaO2 and with signs of enhand exercise toleran. The authors hypothesized that changes in lung mechanics indud by : helium-O2 likely increased stroke volume, Q and systemic _ QO2 . A subsequent study187 went a step further. These authors subdivided COPD patients into dynamic "hyperinflators" and _ "nonhyperinflators," measured quadrips Qm by the ICG _ method98 and calculated QO2m . Helium-O2 increased exercise _ toleran and QO2m in both groups, but at least in part different mechanisms were involved: in hyperinflators, helium-O2 _ increased CaO2 and QO2m in the presen of: no changes in : Q; in nonhyperinflators, helium-O2 improved Q, vascular con_ ductan, and QO2m . These studies nily demonstrate that, also in COPD patients, NIRS can give valuable insights into the mechanisms regulating the integrated responses to conditions of increased O2 demand. Also, COPD patients showed the [deoxy(Hb+Mb)] overshoot during constant work rate exercise142 and a slower reoxygenation kinetics during the recovery following exercise.188,189 Following 6 weeks of high-intensity training, the reoxygenation kinetics became faster, in association with an increased citrate synthase activity and an increased exercise toleran.188 Metabolic Myopathies and Muscular Dystrophies Sin NIRS oxygenation indis reflect skeletal muscle fractional O2 extraction, they appear to be ideally suited to identify and evaluate the impairment of oxidative metabolism in patients with mitochondrial myopathies (MM) and myophosphorylase deficiency (McArdle disease, McA), in whom the incapacity to increase skeletal muscle fractional O2 extraction represents a key pathophysiological mechanism (see the discussion in Grassi et al.110). Following the pioneering work by Bank and Chan,78 Grassi et al.,110 using NIRS, measured [deoxy(Hb+ Mb)] at exhaustion (½deoxyðHb þ MbÞpeak ) during an incremental exercise test to obtain the maximal capacity of skeletal muscle fractional O2 extraction in MM and McA patients. The values were significantly lower than those of controls, and they _ were linearly correlated with VO2p;peak ,110 a close approximation _ of VO2p;max ; they also were inversely related to the time-constant _ of VO2p kinetics,190 another classical variable of functional evaluation of skeletal muscle oxidative metabolism.3,4,10 The most severely impaired patients could not increase their fractional O2 extraction during exercise compared to the values obtained at rest, confirming observations obtained in previous studies by invasive measurements (see the discussion in Grassi et al.110). Moreover, ½deoxyðHb þ MbÞpeak values obtained in MM and McA patients were substantially lower than those obtained in patients with similar signs and symptoms but with a negative biopsy for any known metabolic myopathy.110 In other words, NIRS allowed one to identify and quantify the metabolic impairment in MM and McA patients and could also offer diagnostic clues, for example, in the selection of patients on whom to perform a muscle biopsy. Markedly lower [deoxy(Hb+Mb)] values at submaximal work rates were observed in MM and McA patients.110,140 Journal of Biomedical Optics Lower [deoxy(Hb+Mb)] values at the same submaximal work rate, versus controls, were also observed in MM during incremental handgrip exercise.191 These data suggest an exss in _ _ QO2m for the same VO2m (and work rate), which represents one of the key pathophysiological mechanisms of the diseases. MM and McA patients also showed a marked transient [deoxy(Hb+Mb)] overshoot during constant work rate exercise.140 In McA, the overshoot disappeared in the presen of the "second wind" phenomenon, a sudden decrease in heart rate and perived exertion occurring in these patients after a few minutes of exercise, or during a second exercise preded by a few minutes by a prior exercise.140 The second wind phenomenon is thought to derive from an enhand sympathoadrenal response, which would allow the utilization of glucose deriving from blood or extramuscular sours and of free fatty acids, thereby bypassing the metabolic impairment (the inability to breakdown intramuscular glycogen) at the basis of the disease (see the discussion in Porlli et al.140). In other words, in McA patients, NIRS confirmed the mechanism (sudden enhanment of oxidative metabolism) at the basis of the second wind phenomenon. Another glycolytic defect leading to a decreased capacity to increase fractional O2 extraction during exercise is muscle-type phosphofructokinase (M-PFK) deficiency. The impairment has been demonstrated by NIRS in humans70 and in dogs.192 ½deoxyðHb þ MbÞpeak was also determined by Marzorati et al.193 in a small population of patients with the adult form of Pompe disease (glycogen storage disease type II, due to the deficiency of the lysosomal enzyme acid maltase), before and after 12 months of enzyme replament therapy. Following enzyme replament therapy, the authors observed that ½deoxyðHb þ MbÞpeak slightly increased in some of the patients. In patients with Friedreich's ataxia, the genetic defect decreases the expression of the protein frataxin, localized in mitochondria. The resulting impairment of mitochondrial function and oxidative metabolism has been identified at the skeletal muscle level by 31 P-MRS (see the discussion in Lynch194). These authors observed by NIRS a slower reoxygenation kinetics following exercise, and the degree of impairment of this variable correlated with the length of the triplet GAA repeat, a genetic measure associated with the age of onset of the disease.194 Quaresima and Ferrari195 first investigated by NIRS quadrips muscle oxygenation in patients with Duchenne muscular dystrophy (DMD). An insufficient increase in _ _ QO2m with respect to VO2m occurs in these patients.196 In other words, the functional defect is the opposite of that characterizing MM and McA patients (see above). The dystrophin deficiency results in a loss of sarcolemmal neuronal NO. NO plays a key role in the control of muscle blood flow during exercise, by blunting the vasoconstrictor response to -adrenergic stimulation.123,124 In DMD patients, the impaired NO synthesis would impair the intramuscular matching of _ _ QO2m versus VO2m , leading to an increased O2 extraction, which has indeed been demonstrated by NIRS.196 An enhand O2 extraction has been observed197 also in patients with Becker muscular dystrophy, a milder form of dystrophy in which the intramuscular NO impairment is also present. In these patients, the enhand O2 extraction was associated with the functional impairment. An enhand O2 extraction, on the other hand, has not been described in patients with September 2016 091313-13 Vol. 21(9) Grassi and Quaresima: Near-infrared spectroscopy and skeletal muscle oxidative function. . . limb-girdle muscular dystrophy, in whom neuronal NO synthase is not impaired.196 Caliandro et al.198 could differentiate, by utilizing arterial occlusion testing in postocclusion hyperemia, patients with dermatomyositis and inclusion-body myositis from healthy controls; the differentiation was not possible for patients with polymyositis. A mitochondrial dysfunction, evaluated by the _ recovery kinetics of [PCr] (by 31 P-MRS) and VO2m (by NIRS), was observed in patients with amyotrophic lateral sclerosis.199 Other Diseases Other patients populations characterized by a skeletal muscle oxidative impairment, which was identified by NIRS by the _ analysis of the rate of decrease in VO2m following voluntary or electrically stimulated contractions,94,200 are those with cystic fibrosis201 or spinal cord injuries.202 In a case report, Ryan et al.200 described a threefold increase of the rate of decrease _ in VO2m (indicating a substantial improvement of skeletal muscle oxidative metabolism) in a patient with a spinal cord injury who underwent 12 weeks of enduran training, conducted by neuromuscular electrical stimulation. Malenfant et al.203 observed by NIRS, in patients with pulmonary arterial hypertension, markedly higher [deoxy(Hb+ Mb)] during submaximal exercise at the same absolute work rate versus controls, indicating a markedly higher vastus lateralis fractional O2 extraction. The data suggest an impaired _ QO2m in these patients, which would be compatible with the lower capillary density observed in the same muscle of the other leg. The data would also be compatible with the increases in endothelin-1 levels and with the decreases in NO and prostaglandins. Interestingly, hyperoxic breathing, while normalizing SO2a , did not substantially affect the [deoxy(Hb+Mb)] _ response, confirming an impairment in QO2m deriving from the structural and functional impairments mentioned above. In patients with fibromyalgia, Shang et al.204 combined the utilization of a commercially available NIR oximeter with a custom-built NIR diffuse correlation spectroscopy (DCS) instru_ ment for tissue Qm measurements. Experiments were conducted during fatiguing knee extensors isometric contractions and a forearm arterial occlusion protocol. From the O2 extraction _ _ and Qm data, VO2m was calculated (see also below). The patients showed, versus the controls, a lower fractional O2 extraction and a slower reoxygenation kinetics. Muscle NIRS measurement is an important tool in identifying high-risk patients in septic and hemorrhagic shock, by offering a continuous and noninvasive monitoring of tissue hypoperfusion.205,206 According to these authors, during active shock resuscitation changes in skeletal muscle oxygenation levels are correlated with changes in O2 delivery, base deficit, and blood lactate levels. A drop in muscle oxygenation levels predicts death and multiorgan failure, and may be helpful in making critical clinical decisions. Instrumentation Advanments The present section is aimed at briefly reporting the advanments made in the last few years in the field of NIRS instrumentations suitable for muscle studies and at mentioning the future directions of the development/utilization of these NIRS devis. The main commercial NIRS oximeters for muscle studies, with the related key features and measurable parameters, are listed in Table 1. Sin 2011 (when a homologous table was Journal of Biomedical Optics published in the review by Ferrari et al.24), different batteryoperated portable/wireless muscle oximeters have been commercialized. The CW-NIRS portable/wearable systems, with wireless data transmission, are reported in Table 2. The details about each system can be found in the related company's websites. Three different NIRS techniques are utilized in the field of muscle oximetry. Each of them is based on a specific type of illumination: (1) the most commonly used CW instruments, based on constant illumination of the tissue, simply measure the attenuation of light through the tissues; (2) FD instruments, illuminating the tissues with intensity-modulated light, measure both the attenuation and the phase delay of the emerging light; and (3) TD instruments, illuminating the tissue with short pulses of light (few picoseconds), detect the temporal broadening of the pulse after propagation through the tissues. The quantitation of the NIRS measurable parameters depends on the adopted NIRS technology. Different NIRS methods using the CW modality have been developed to measure muscle O2 saturation ( , %). The measurement assures an accurate quantitation of the oxygenation changes occurring at the skeletal muscle level. The CW-based systems offer the advantages of low-cost and easy transportability. In asnding order, CW-, FD-, and TD-based instruments require increased cost and technological complexity. On the other hand, FD and TD techniques offer a better depth discrimination than that achieved by CW instruments and allow the absolute characterization of the tissue optical properties (absorption and redud scattering coefficients), from which it is possible to retrieve absolute conntration values of [oxy(Hb+ Mb)] and [deoxy(Hb+Mb)], and the derived parameter [total(Hb+Mb)]. The available CW-NIRS instruments do not determine redud scattering coefficient and circumvent the problem by assuming that the coefficient is substantially constant during rest and exercise. This assumption has been criticized.207 The issue has also been discussed in the review by Ferrari et al.24 Most of the NIRS instruments reported in Table 1 are represented by two to six channels and CW brain oximeters utilized also for physiological/pathophysiological muscle studies. These oximeters are the most commonly utilized NIRS instruments because they are of relatively low/moderate cost and transportable. Muscle NIRS instrumentations are characterized by a relatively high temporal resolution; the sampling rate is up to 100 Hz in some CW systems, 50 Hz in the only commercial FD system, and 0.2 Hz in the first TD oximeter with mark, commercialized in 2015 (Table 1). An original muscle oximeter has been very rently introdud by Reflectan Medical; the instrument collects muscle spectra and calculates as well as pHm . The sector of wearable health technology is gaining increasing interest. The use of low cost wearable devis or wearable biosensors, which allow a constant monitoring of physiological signals, is essential for the advanment of both the diagnosis and the treatment of diseases, as well as for monitoring active life style. Very rently, a textile NIRS system has been utilized for monitoring arterial O2 saturation (measured by pulse oximetry) and muscle oxygenation changes.208 Wearable wireless fingertip pulse oximetry devis, for arterial O2 saturation measurement, are already available for smart phones. In addition, a wireless and smartphone controllable NIRS system (DynaSense September 2016 091313-14 Vol. 21(9) Grassi and Quaresima: Near-infrared spectroscopy and skeletal muscle oxidative function. . . Inc., Japan) has been very rently presented.209 Table 2 includes also two wireless systems that are waterproof, then allowing muscle oxygenation measurements in swimmers.210 For a better understanding of skeletal muscle physiology, muscle oxygenation data should be ideally integrated with regional blood flow measurements. Direct and indirect NIRS _ methods to measure Qm 20,96 have been discussed above. A novel technique, known as DCS, has been rently developed for quantifying relative changes in microvascular regional blood flow. Basically, DCS uses coherent NIR light to penetrate deep tissues and measures speckle fluctuations of the diffuse light that are sensitive to the motions of red blood lls in tissues.211,212 The well validated DCS provides a portable, noninvasive, and inexpensive alternative for measuring microvascular blood flow. A rent review article has described the _ progresses made in the field of DCS for assessing Qm during 213 dynamic exercise. This technology allows also the estimation of skeletal muscle oxygenation levels. So far, two DCS-NIRS systems have already been commercially available: FloMo (Hemophotonics S.L., Spain) and MetaOx (ISS, _ United States). The former measures only Qm, while the latest provides concomitant measurements of and absolute [oxy (Hb+Mb)] and [deoxy(Hb+Mb)] by FD-NIRS. The method has been utilized on the head of newborns and on the skeletal muscles of adults. Giovannella et al.214 have integrated the -FloMo with a TD-NIRS prototype (TRS20, Hamamatsu _ Photonics, Japan) to measure Qm and absolute [total(Hb+ Mb)] during quadrips exercise. A different approach for meas_ uring Qm has been utilized by Ornim Inc. (United States), which has developed the brain/muscle oximeter "rOx 3210F" using NIRS and weak acoustic beams to identify light emerging from deep tissue layers. Many sophisticated multichannel CW-NIRS instrumentations, dedicated to brain cortex two-dimensional (2-D) imaging (topography), are commercially available and largely utilized in different areas in the field of neuroscien.25,215 Unfortunately, the probes they are equipped with cannot be utilized for skeletal muscle 2-D imaging studies. In 2015, an eight-measurement point wireless system for 2-D brain/muscle measurements has been commercialized (Table 2). In the framework of a National Aeronautics and Spa Administration (NASA) project, a 64-measurement point wireless NIRS system has been developed and integrated with simultaneous electrocardiography and actigraphy monitoring, in order to record 2-D muscle imaging data up to several days.216 Although a threedimensional (3-D) NIR imaging (tomography) of the human forearm, based on TD techniques, had been proposed217 almost 15 years ago, no further consistent progresses in this field have been made. Rently, cross-sectional 2-D images of the foot [total(Hb+Mb)], using a multichannel 3-D CW system during thigh cuff occlusion, were reported.218 The state-of-the-art of TD-NIRS has been rently reviewed.219 Many progresses have been done in the last 2 years that open a new way to compact, low-cost, and highperforman TD devis for diffuse muscle optical imaging and spectroscopy. The new frontiers in TD diffuse optics are covered by an article of this Special Section. 220 Briefly, consistent progresses are expected conrning noncontact scanning 2-D imaging systems for TD-NIRS based on a null sour­ detector separation.221­223 These systems, already tested in the human forearm, utilize new fast detectors for extreme depth penetration and sensitivity.224,225 Journal of Biomedical Optics Conclusions Researchers should always be critical and careful about the technologies and methods they utilize, but within reasonable limits. Many exercise physiologists, in our opinion, have been exssively critical about NIRS in the past. The method has important limitations but presents significant advantages compared to other methods. Skeletal muscle fractional O2 extraction, the main variable that can be noninvasively evaluated by NIRS, can yield relevant information on key physiological and pathophysiological mechanisms, relevant in the evaluation of exercise performan and exercise toleran in healthy subjects and in patients. To put the method in a broader perspective it should be recognized, for example, that the BOLD phenomenon,226 as obtained by functional magnetic resonan imaging, a functional neuroimaging technique which has profoundly revolutionized neuroscien with applications ranging from normal brain development, aging, brain disorders and diseases, is conptually very similar to the fractional O2 extraction variable determined by NIRS in skeletal muscle. Both variables, indeed, substantially reflect changes in tissue oxygenation resulting from the dynamic balan between tissue O2 consumption and O2 supply. The rent diffusion of apparently easy-to-use portable NIRS instruments had to the proliferation of studies, sometimes of dubious quality. An example is represented by studies, which claim to obtain absolute oxygenation indis when the instrumentation they employ does not allow this. The authors must be aware of the limitations of the method and should always have a clear and sound hypothesis to test. However, in the right hands (a good physiological background is mandatory), NIRS can offer insights into key physiological and pathophysiological adaptations to conditions of increased O2 needs, often involving, in an integrated manner, different organs of the body. In terms of patients' evaluation, NIRS allows to determine the evolution of functional impairments of skeletal muscle oxidative metabolism with time, to identify correlations with clinical symptoms, to evaluate the effects of therapeutic or rehabilitative interventions, and to gain pathophysiological and diagnostic insights. 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Ferrari, "Assessment of quadrips oxygenation in patients with myopathies by near infrared spectroscopy," Neurology 51, 1238­1239 (1998). 196. M. Sander et al., "Functional muscle ischemia in neuronal nitric oxide synthase-deficient skeletal muscle of children with Duchenne muscular dystrophy," Proc. Natl. Acad. Sci. 97, 13818­13823 (2000). 197. E. Allart et al., "Evaluation of muscle oxygenation by near-infrared spectroscopy in patients with Becker muscular dystrophy," Neuromuscul. Disord. 22, 720­727 (2012). 198. P. Caliandro et al., "Idiopathic inflammatory myopathies evaluated by near-infrared spectroscopy," Muscle Nerve 51, 830­837 (2015). 199. T. E. Ryan et al., "Skeletal muscle oxidative capacity in amyotrophic lateral sclerosis," Muscle Nerve 50, 767­774 (2014). 200. T. E. Ryan et al., "Case report: enduran electrical stimulation training improves skeletal muscle oxidative capacity in chronic spinal cord injury," Arch. Phys. Med. Rehabil. 94, 2559­2561 (2013). 201. M. L. Erickson et al., "Skeletal muscle oxidative capacity in patients with cystic fibrosis," Exp. Physiol. 100, 545­552 (2015). 202. M. L. Erickson et al., "Near-infrared assessment of skeletal muscle oxidative capacity in persons with spinal cord injury," Eur. J. Appl. Physiol. 113, 2275­2283 (2013). 203. S. Malenfant et al., "Impaired skeletal muscle oxygenation and exercise toleran in pulmonary hypertension," Med. Sci. Sports Exercise 47, 2273­2282 (2015). 204. Y. Shang et al., "Noninvasive optical characterization of muscle blood flow, oxygenation and metabolism in women with fibromyalgia," Arthritis Res. Ther. 14: R236 (2012). 205. R. J. Santora and F. A. Moore, "Monitoring trauma and intensive care unit resuscitation with tissue hemoglobin oxygen saturation," Crit. Care 13(Suppl. 5), S10 (2009). 206. B. R. Soller et al. "Noninvasively determined muscle oxygen saturation is an early indicator of ntral hypovolemia in humans," J. Appl. Physiol. 104, 475­481 (2008). 207. L. F. Ferreira, D. M. Huebner, and T. J. Barstow, "Effects of assuming constant optimal scattering on measurements of muscle oxygenation by near-infrared spectroscopy during exercise," J. Appl. Physiol. 102, 358­367 (2007). 208. M. Krehel et al., "Development of a luminous textile for reflective pulse oximetry measurements," Biomed. Opt. Express 5, 2537­2547 (2014). 209. T. Watanabe et al., "Development of portable, wireless and smartphone controllable near-infrared spectroscopy system," in 43rd Annual Meeting of the Int. Society on Oxygen Transport to Tissue (ISOTT), Wuhan, China, July 11­16, 2015, Poster n. 6. (2015). 210. B. Jones, M. Dat, and C. E. Cooper, "Underwater near-infrared spectroscopy measurements of muscle oxygenation: laboratory validation and preliminary observations in swimmers and triathletes," J. Biomed. Opt. 19, 127002 (2014). 211. T. Durduran and A. G. Yodh, "Diffuse correlation spectroscopy for non-invasive, micro-vascular rebral blood flow measurement," Neuroimage 85, 51­63 (2014). 212. G. Yu et al., "Near-infrared diffuse correlation spectroscopy for assessment of tissue blood flow," in Handbook of Biomedical Optics, Chapter 10, pp. 195­216, CRC Press, Boca Raton, Florida (2011). 213. Y. Shang, K. Gurley, and G. Yu, "Diffuse correlation spectroscopy (DCS) for assessment of tissue blood flow in skeletal muscle: rent progress," Anat. Physiol. 3, 128 (2013). 214. M. Giovannella et al., "Hybridization of Hamamatsu TRS-20 timeresolved near-infrared spectroscopy and HemoPhotonics HemoFloMo diffuse correlation spectroscopy systems," in 2015 European Conf. on Biomedical Optics, Advans in Instrumentation and Technology II, Munich, Germany, 21­25 June 2015, Abstract 9538­23 (2015). 215. F. Scholkmann et al., "A review on continuous wave functional nearinfrared spectroscopy and imaging instrumentation and methodology," Neuroimage 85(Pt 1), 6­27 (2014). 216. Q. Zhang et al., "Twenty-four-hour ambulatory recording of rebral hemodynamics, systemic hemodynamics, electrocardiography, and actigraphy during people's daily activities," J. Biomed. Opt. 19, 047003 (2014). 217. E. M. Hillman et al., "Time resolved optical tomography of the human forearm," Phys. Med. Biol. 46, 1117­1130 (2001). 218. M. A. Khalil et al., "Detection of peripheral arterial disease within the foot using vascular optical tomographic imaging: a clinical pilot study," Eur. J. Vasc. Endovasc. Surg. 49, 83­89 (2015). 219. A. Torrilli et al., "Time domain functional NIRS imaging for human brain mapping," Neuroimage 85, 28­50 (2014). 220. A. Pifferi et al., "New frontiers in time-domain diffuse optics," J. Biomed. Opt. 21(9), 091310 (2016). 221. D. Contini et al., "Effects of time-gated detection in diffuse optical imaging at short sour-detector separation," J. Phys. D: Appl. Phys. 48, 045401 (2015). 222. M. Mazurenka et al., "Non-contact in vivo diffuse optical imaging using a time-gated scanning system," Biomed. Opt. Express 4, 2257 (2013). 223. H. Wabnitz et al., "Time-domain diffuse optical imaging of tissue by non-contact scanning," in Advand Time-Correlated Single Photon Counting Applications, Springer Series in Chemical Physics, Vol. 111, pp. 561­585, Springer International Publishing, Switzerland (2015). 224. A. Dalla Mora et al., "Towards next-generation time-domain diffuse optics for extreme depth penetration and sensitivity," Biomed. Opt. Express 6, 1749­1760 (2015). 225. A. Dalla Mora et al., "Fast silicon photomultiplier improves signal harvesting and redus complexity in time domain diffuse optics," Opt. Express 23, 13937 (2015). 226. C. I. Mark, E. L. Mazerolle, and J. J. Chen, "Metabolic and vascular origins of the BOLD effect: implications for imaging pathology and resting-state brain function," J. Magn. Reson. Imaging 42, 231­246 (2015). Bruno Grassi, reived his MD and PhD degrees and now is a professor of physiology in the Medical School of the University of Udine (Italy), where he is head of the Exercise Physiology Laboratory. His research interests are in the fields of exercise and environmental physiology, skeletal muscle bioenergetics, with special referen to oxidative metabolism, in health and disease. He is the author of 112 scientific publications on international peer-reviewed journals and of 25 chapters in books. Valentina Quaresima reived her PhD and now is an associate professor of biochemistry at L'Aquila University, Italy. She has pioneered clinical applications of NpIRS of the adult and infant muscle/brain and has given substantial contributions to wider NIRS scientific community. Sin 1993, she has been involved in the study of localized measurements of muscular and rebral oxygenation in humans by different NIRS techniques. She is the author of 100 scientific publications on international peer-reviewed journals. Journal of Biomedical Optics 091313-20 September 2016 Vol. 21(9) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biomedical Optics SPIE

Near-infrared spectroscopy and skeletal muscle oxidative function in vivo in health and disease: a review from an exercise physiology perspective

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SPIE
Copyright
© 2016 Society of Photo-Optical Instrumentation Engineers (SPIE)
Subject
Special Section on Clinical Near-Infrared Spectroscopy and Imaging; Paper
ISSN
1083-3668
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1560-2281
DOI
10.1117/1.JBO.21.9.091313
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27443955
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Abstract

In most daily activities related to work or leisure, the energy for muscle work substantially comes from oxidative metabolism. Functional limitations or impairments of this metabolism can significantly affect exercise toleran and performan. As a method for the functional evaluation of skeletal muscle oxidative metabolism, near-infrared spectroscopy (NIRS) has important strengths but also several limitations, some of which have been overcome by rent technological developments. Skeletal muscle fractional O2 extraction, the main variable which can be noninvasively evaluated by NIRS, is the result of the dynamic balan between O2 utilization and O2 delivery; it can yield relevant information on key physiological and pathophysiological mechanisms, relevant in the evaluation of exercise performan and exercise toleran in healthy subjects (in normal and in altered environmental conditions) and in patients. In the right hands, NIRS can offer insights into the physiological and pathophysiological adaptations to conditions of increased O2 needs that involve, in an integrated manner, different organs and systems of the body. In terms of patient evaluation, NIRS allows determination of the evolution of the functional impairments, to identify their correlations with clinical symptoms, to evaluate the effects of therapeutic or rehabilitative interventions, and to gain pathophysiological and diagnostic insights. © 2016 Society of Photo-Optical Instrumentation Engineers (SPIE) [DOI: 10.1117/1.JBO.21.9.091313] Keywords: near-infrared spectroscopy; skeletal muscle bioenergetics; skeletal muscle oxidative metabolism; exercise physiology; exercise toleran; review. Paper 150870SSVR reived Jan. 6, 2016; acpted for publication Jun. 28, 2016; published online Jul. 21, 2016. Introduction Near-infrared (NIR) spectroscopy (NIRS) applied to skeletal muscle has often been seen with some skepticism by many exercise physiologists. On at a scientific meeting, one of the authors of the present review was told by Dr. Bengt Saltin (one of the most influential exercise physiologists of the last decades): "Unless you manage to measure O2 consumption, this technique has not much physiological interest." We respectfully disagree with Dr. Saltin's statement. Although we recognize that NIRS has several limitations, some of which have been overcome by rent technological developments, the method also has important strengths and can give valuable (and noninvasive) functional insights into skeletal muscle oxidative metabolism in vivo during exercise, in health and disease. At this purpose, the present review has been devoted, from an "exercise physiology point of view," to discuss some of the main issues related with the role of NIRS in the functional assessment of oxidative metabolism in skeletal muscles during exercise, with specific attention to integrative aspects and to the factors limiting exercise toleran. Attention will also be paid to studies carried out in diseased populations, as well as to some rent and exciting technical and methodological developments. Oxidative Metabolism During Exercise and Tools of Investigation *Address corresponden to: Bruno Grassi, E-mail: bruno.grassi@uniud.it During supramaximal exercise lasting up to a few seconds, the metabolic power output in humans can increase by about 150 to 200 times compared with the value observed at rest. Among the tissues of the body, only skeletal muscle can sustain such extraordinary increases in metabolic power output, which is made possible by the splitting of high energy phosphates present in muscle as adenosine triphosphate (ATP) and creatine phosphate (PCr). If the exercise is carried out for longer periods of time (up to several minutes, or even hours), the maximal metabolic power output, which can be sustained, decreases hyperbolically as a function of the duration of the exercise1­4 and oxidative metabolism becomes the prevalent, or substantially the only, mechanism responsible for ATP resynthesis. In other words, in most of our daily activities, either related to work or leisure, the energy for muscle work substantially comes from the oxidation of glucose and lipids in muscle fibers, culminating in oxidative phosphorylation at the mitochondrial respiratory chain. It is not surprising, then, that the maximal power by oxidative metabolism and the fraction of this power, which can be sustained for relatively long periods of time, are intrinsically related to exercise performan and toleran. An impairment of September 2016 Journal of Biomedical Optics 091313-1 Vol. 21(9) Grassi and Quaresima: Near-infrared spectroscopy and skeletal muscle oxidative function. . . oxidative metabolism, which occurs in several types of patients, inevitably leads to a redud exercise toleran, which may represent one of the main determinants of the clinical picture and quality of life, as well as an important predictor of mortality.5 The availability of reliable tools to investigate in vivo, with precision and reproducibility, muscle oxidative metabolism, possibly noninvasively and with a reasonably elevated temporal resolution, appears therefore of utmost importan. This applies to athletes (sports activities), healthy subjects (both in normal and in altered environmental conditions), and patients affected by many diseases. In the patients, these tools would allow the follow-up of the impairments with time, the identification of potential correlations with clinical symptoms, the evaluation of the effects of therapeutic or rehabilitative interventions, and could also yield pathophysiological and diagnostic insights. _ Whole body oxygen consumption (VO2 ) can be measured with relatively good precision (variability of measurements of about 5% in the best laboratories) at the mouth of the subject _ _ or at the alveolar level6 (pulmonary VO2, VO2p ) by the classic open circuit method. These measurements have been carried out in innumerable studies and lead to the definition of conpts like _ the maximal aerobic power (VO2p;max ), which evaluates the integrated performan of the respiratory, cardiovascular, and skeletal muscle components of the O2 pathway from ambient air to the mitochondria of skeletal muscles. The availability of _ methods to determine VO2p with an elevated temporal resolution ("breath-by-breath" measurements)6­8 expanded the spectrum of the functional evaluation variables and allowed the introduction of conpts such as the "gas exchange threshold,"9 the "critical _ power,"1 or the various phases of the VO2 "kinetics."3,4,6,10 The _ main limitation of the whole body VO2p measurements resides in the impossibility to discriminate between the exercising muscles and the rest of the body, as well as between different muscles engaged in the exercise. Moreover, the presen of O2 stores between the site of measurement (the mouth or the alveoli) and the sites of gas exchange at the skeletal muscle level complicates data interpretation during metabolic transitions.6 A further limitation derives from the diffusion of automated and apparently easy-to-use "metabolimeters," which to the proliferation of data and studies of dubious quality. _ The relationship between VO2 , blood flow and O2 extraction can be expressed by the Fick equation, based on the principle of mass conservation. If applied "across" a single muscle or a single muscle group, the equation reads as follows: EQ-TARGET;temp:intralink-;e001;63;258 from the exercising muscle, with a resulting "contamination" of venous blood coming from other muscles. Alternative methods allowing a functional evaluation in vivo of skeletal muscle oxidative metabolism derive from the utilizations of phosphorus (31 P) or proton (1 H) nuclear magnetic resonan spectroscopy (31 P-MRS, and 1 H-MRS, respectively). As far as 31 P-MRS, the method has been utilized to determine the kinetics of recovery of muscle [PCr] following exercise. After assuming the equilibrium of the creatine kinase (CK) reaction, the rate of recovery of [PCr] is a function of mitochondrial ATP production, and therefore it can be considered a tool for evaluating the function of skeletal muscle oxidative metabolism.15 1 H-MRS, on the other hand, detects myoglobin (Mb) desaturation, thereby allowing an estimation of intrallular PO2.16,17 These technologically formidable methods are intrinsically limited by the very high cost of the instrumentation and by the strict constraints on the exercise modality determined by the size of the bore of the magnet. In short, the above mentioned methods, although widely utilized, have intrinsic limitations. Therefore, noninvasive, precise, reproducible, and relatively low-cost functional evaluation tools to be utilized in vivo, characterized by an elevated temporal resolution, allowing the discrimination between (and possibly within) skeletal muscle groups, would be needed. Does NIRS satisfy, at least in part, these needs? The following sections of the present review will deal with this issue. Near-Infrared Spectroscopy from an Exercise Physiology Point of View _ _ VO2m ¼ Qm à ½Cða-vÞO2m ; (1) _ _ _ in which VO2m represents muscle VO2 , Qm muscle blood flow, and Cða-vÞO2m arterial-venous O2 conntration differen across the muscle. By rearranging Eq. (1): EQ-TARGET;temp:intralink-;e002;63;181 Several exllent review articles about the application of NIRS in skeletal muscle have been published.18­25 These reviews carry the relevant information on principles, methods, instruments, and so on. Ferrari et al.24 included a useful list of practical recommendations. The present review article has been specifically devoted to discuss, from an "exercise physiology point of view," some of the main issues related with the role of NIRS in the functional evaluation in vivo of oxidative metabolism in skeletal muscles, within an integrated perspective related to exercise toleran. A specific attention will be paid to NIRS studies carried out in diseased populations, as well as to some rent and exciting technical and methodological developments. The latter testify how NIRS, as a tool of functional investigation of skeletal muscles, is still in a steep portion of a gain in knowge versus time curve. The role of NIRS in evaluating oxidative skeletal muscle performan in sport activities and in athletes, which would deserve a dedicated review article, is not covered in the present article. Likewise, the effects of exercise on brain hemodynamics, as measured by NIRS,26,27 as well as the role of NIRS in the evaluation of brain function21,25 are not discussed. Cða-vÞO2m _ _ ¼ VO2m Qm : (2) Tissue Interrogated by the Near-Infrared Spectroscopy Probe _ VO2m measurements have been carried out with a high temporal resolution also during metabolic transitions in humans11,12 or in isolated in situ animal models.13,14 The main limitations of this approach can be summarized as follows: invasiveness of the measurements; technical problems inherently associated with in vivo blood flow measurements; the substantial impossibility (in human studies) to sample venous blood directly effluent Journal of Biomedical Optics When the probe is applied on the skin overlying a muscle of interest, NIRS instruments can interrogate only a relatively small (2 to 6 cm3 ) and superficial volume of skeletal muscle tissue.28 It is generally acpted that the depth of penetration of the NIR light in tissues roughly corresponds to half of the distan between the light sour and the detector (which is usually between 3 and 5 cm). This intrinsic technical limitation makes the measurement of the thickness of the skin and September 2016 091313-2 Vol. 21(9) Grassi and Quaresima: Near-infrared spectroscopy and skeletal muscle oxidative function. . . subcutaneous adipose tissue layer, at the site of plament of the NIRS probe, mandatory. The measurement can be carried out by a caliper or, more precisely, by ultrasound.29,30 The rent availability of low-cost hand-held ultrasound devis has significantly facilitated these measurements. Although no cutoff values are recognized as standard, a value greater than 20 mm would presumably make NIRS measurements, as carried out by standard instruments, rather meaningless in terms of investigating skeletal muscle. This precludes the utilization of the method in subjects with a relatively thick layer of subcutaneous fat, such as obese patients or in patients with significant muscle atrophy. Table 1 Main commercial muscle NIRS oximeters. Devi Oxymon Mk IIIa Company, country Artinis, The Netherlands Astem, Japan Technique Multidistan CW Multidistan CW Multidistan CW Type of sours N. of wl 2 Timeresolution No. of Measurable (Hz) channels parameters Approvals 250 2 Websites www.artinis.com Exercise Monitor Hb11b www.astem-jp.com Fore-sight Elite®a CAS, Inc., United States NIRO-200 NXa , www.casmed.com Hamamatsu, Multidistan Japan CW Hamamatsu, TD Japan ISS, United States Masimo, United States Multidistan FD Multidistan CW www.hamamatsu.com tNIRS-1a , atHb www.hamamatsu.com OxiplexTSTMa , atHb www.iss.com O3TM Regional Oximetrya up to 6 , SO2p www.masimo.com INVOSTM 5100Ca Medtronic, United States moorVMS-NIRS Multidistan CW www.medtronic.com Moor, United Multidistan Kingdom CW Nirox, Italy Multidistan CW Multidistan CW www.moor.co.uk NIMOab www.nirox.it SenSmartTM Model X-100a Nonin, United States Omegawave, Japan Ornim, Inc., United States up to 5 , SO2p www.nonin.com BOM-L1 TR SF Single distan www.omegawave.co.jp roxTM 3210Fa Multidistan CW , flowc www.ornim.com CareGuideTM 4100 Reflectan, Multidistan Med., United CW States Multiple , pHm www.reflectanmedical. com Note: atHb, absolute total hemoglobin conntration; , European conformity marking as a medical devi; CW, continuous-wave spectroscopy; FD, frequency-domain spectroscopy; , Food and Drug Administration's approval; , light emitting diodes; N., number; pHm, muscle pH; , muscle oxyhemoglobin saturation; SO2p , peripheral arterial oxyhemoglobin saturation (measured by pulse oximetry); TD, time-domain spectroscopy; wl, wavelengths. a Brain oximeter utilized also in muscle studies b Oximeter with fat-layer compensation c Cortical microcirculation blood flow measurement using Doppler shift in coherent light signals. Journal of Biomedical Optics 091313-3 September 2016 Vol. 21(9) Grassi and Quaresima: Near-infrared spectroscopy and skeletal muscle oxidative function. . . The fact that the NIR light has to cross the skin and subcutaneous fat in order to reach the underlying skeletal muscle tissue inevitably causes a sensitivity problem and represents a "contamination" of the signal of interest, which is the one coming from skeletal muscle. The problem can be particularly significant in conditions in which skin blood flow increases during exercise for thermoregulatory purposes (see also below). Several "correction algorithms" have been proposed (see the discussion in Ferrari et al.24), which, however, are not included in most of the commercial instruments (Tables 1 and 2). Ohmae et al.31 have rently described a simple method, which does not require ultrasound, for the sensitivity correction for the influen of fat thickness on muscle oxygenation, based on the estimation of fat thickness by time-domain NIRS (TD-NIRS) (see below). van Beekvelt et al.32 and Koga et al.29 proposed other correction _ algorithms based on the assumption that resting VO2m [estimated on the basis of the conntration increases in deoxy-hemoglobin (Hb) and deoxy-myoglobin (Mb), [deoxy(Hb+Mb)]) after applying a cuff occluding the arterial circulation, see below] would be inversely correlated with skin + subcutaneous tissue thickness. Another approach, based on [total(Hb+Mb)] values obtained at rest by quantitative multichannel TD-NIRS, has been proposed.33 In the quadrips muscle (often investigated by NIRS studies) and in other muscles as well, deeper muscle regions are characterized by a greater proportion of oxidative fibers compared to superficial regions34 and by a more oxidative energy metabolism. Accordingly, the deeper fibers have a greater sensitivity toward vasodilatory control mechanisms.35 An anatomical "gradient" in fiber type is presumably associated with a gradient also in terms of metabolism and blood perfusion.36 This has been substantially confirmed, in the quadrips muscle of humans, in studies carried out by utilizing a "high-power" TD-NIRS instrument,37,38 which allows interrogation of slightly deeper portions of muscle. This instrument, which delivers NIR light with a power about 30 times higher than that of traditional NIRS instruments, allows an effective mean penetration depth of about 3 cm and may represent a substantial technical improvement. Another problem related to the small volume of tissue interrogated by conventional NIRS instruments derives from the fact that muscle activation and metabolism,39 and even more markedly so blood perfusion36,40,41 are heterogeneously distributed within and between exercising muscles. Exllent reviews on metabolic and blood flow heterogeneities in skeletal muscle, in health and disease, have been rently published.42,43 Sin skeletal muscle fractional O2 extraction (see below) can increase during exercise only three to four times above the values at rest, _ whereas VO2m can increase 100-fold, directing blood flow according to O2 needs is crucial to allow sustained contractile performan.43 Most studies (but not all44) found good matching between _ _ muscle O2 delivery (QO2m ¼ Qm à CaO2 , in which CaO2 rep_ resents the arterial O2 content), or substrates delivery, and VO2m between macroscopic (volume of several cm3 ) regions of a muscle, or between different muscles involved in the exercise.30,45,46 However, when the resolution capacity of the measurements went down to about 1 cm3 , the spatial matching _ _ between QO2m and VO2m (assessed via PCr depletion) was far from being perfect.45 The presen (or absen) of a matching between the two variables could be more functionally relevant at a microscopic level. According to Segal,47 the recruitment of contiguous "microvascular units," whose volume is orders of magnitude smaller than the volume investigated by NIRS, could be difficult to match with the recruitment of motor units, whose fibers are sparse within the muscle. In other words, blood flow cannot specifically increase to a specific muscle fiber or to the fibers of a motor unit,47 but must rather Table 2 Main commercial continuous wave portable/wearable NIRS systems for muscle studies with wireless data transmission. Devi OctaMon Company, country Artinis, The Netherlands Technique Singistan Multidistan n.a. N. of wl 2 Timeresolution (Hz) 50 No. of channels 8 Measurable parameters O2 HbHHb changes , LT Websites www.artinis.com PortaMon Mk IIb BSX Insight 2.0 Artinis, The Netherlands BSX Athletics, LLC, United States DynaSense Inc., Japan 2 n.a. 10 n.a. www.artinis.com www.bsxinsight.com PocketNIRS Duo®c,d Moxye Singistan Monte Carlo modeling Multidistan Multidistan O2 HbHHb changes www.dynasense.co.jp Moxy Monitor, United States Nirox, Italy Pathonix Innovation Inc., Canada www.moxymonitor. com www.nirox.it www.pathonix.com MyNimo OxiTor-M2 , atHb Note: atHb, absolute total hemoglobin conntration; HHb, deoxyhemoglobin; LT, lactate threshold evaluation by tracing; n.a., not available; N., number; O2 Hb, oxyhemoglobin; , muscle oxyhemoglobin saturation; wl, wavelengths. a Muscle imager. b Aclerometer is available on request. c Commercially available only in Japan. d Smartphone controllable system. e Water resistant case. Journal of Biomedical Optics 091313-4 September 2016 Vol. 21(9) Grassi and Quaresima: Near-infrared spectroscopy and skeletal muscle oxidative function. . . increase over a relatively wide region of the muscle, leading to the possibility of "over-perfusing" inactive or relatively inactive fibers. _ In any case, the apparently good matching between QO2m _ and VO2m in different macroscopic areas of the same muscle, or of different muscles,45,46,48 appears to be difficult to reconcile with the macroscopic heterogeneity of muscle deoxygenation observed by several studies.29,37,49 The issue, in our opinion, is not sett, and further studies with higher spatial resolution devis are needed. In summary, the area of muscle investigated by the NIRS probe may not represent, in terms of fiber types, fiber activation : _ and the matching of VO2m and QO2m , a reliable picture of the situation in the whole muscle. In this respect, however, it should be considered that the same limitation, frequently overlooked, is intrinsically associated with other diffusely utilized methods, which investigate only a small and relatively superficial portion of a muscle, such as muscle biopsy. Where do the Near-Infrared Spectroscopy Signals Come From? The NIRS signals are the result of the weighted average of the O2 saturations of the heme groups of Hb in the vascular bed (small arteries, arterioles, capillaries, venules, small veins) and of the Mb heme group in muscle fibers. In terms of Hb, most of the signal comes from small vessels, because larger arteries and veins (greater than 1 mm in diameter) have very high heme conntrations, which absorb all the NIR light. Although it is often considered that venous­venular compartments represent the majority of blood present in skeletal muscle,19,20 direct measurements suggest that this may not be the case. According to Poole and Mathieu-Costello,50 capillaries would contribute to >90% of the total blood volume in muscle. According to these data, the storage of blood in veins would mainly occur externally to the muscle. Thus, the vascular component of the NIRS signals would predominantly come from the capillaries. Sin in normal conditions, all regions of the muscle reive nearly-fully oxygenated arterial blood, oxygenation changes detected by NIRS would mainly reflect changes in capillary (Hb-related) and intrallular (Mb-related) O2 levels. A relative controversy exists about the role played in NIRS signals by the heme group in Mb. It has been traditionally assumed that Hb is responsible for most of the overall NIRS signal.51,52 The conpt is supported by the observation of strong correlations, observed in different experimental models, between muscle oxygenation values obtained by NIRS and O2 saturation in venous blood (see below). Studies carried out by NIRS in combination with 1 H-MRS, aimed at distinguishing between Hb and Mb desaturation during ischemia and exercise, yielded conflicting results.53,54 According to modeling papers,55,56 Mb may contribute to 50% of the total [Hb+Mb] NIRS signal. This conpt is supported by a study in which the contribution of Hb and Mb to in vivo optical spectra was determined by wavelength shift analysis.57 The relative role of Hb versus Mb is likely different at rest and during exercise. As discussed above, most of the intravascular NIRS signals likely come from the capillaries. In resting conditions, capillary hematocrit can be significantly lower than that in the systemic circulation.58 During exercise, on the other hand, capillary hematocrit increases, reaching values that are not substantially different from those in larger vessels.58 Thus, the Journal of Biomedical Optics role of Hb (versus that of Mb) in determining the NIRS signals would be higher during exercise compared to rest. The possibility to separate Hb and Mb desaturation could give valuable insights into the mechanisms regulating peripheral _ O2 diffusion and VO2m . For example, by applying NIRS on a rat model of isolated hindlimb, perfused with Hb-free welloxygenated Krebs-Henseleit buffer, Takakura et al.59 isolated the signal deriving from Mb desaturation and could calculate the intrallular PO2, making inferens on the role of Mb in O2 supply to mitochondria at exercise onset. In any case, the whole diffusion pathway, including vascular Hb and intrallular Mb, would desaturate during exercise with a similar time course.56 Therefore, a greater contribution of Mb than Hb to the NIRS [deoxy(Hb+Mb)] signal would not invalidate the interpretation that changes in this signal reflect frac_ tional O2 extraction, the dynamic balan between VO2m and _ QO2m in the volume of tissue under consideration.56 This would apply even if a separation between the Hb and the Mb signal is not feasible or is not carried out. In the next sections of the manuscript, we will discuss why, in our opinion, fractional O2 extraction is by itself of interest. In terms of the intrallular signals detectable by NIRS, cytochrome oxidase can also absorb NIR light. However, there is substantial agreement that assessment of the redox state of mitochondrial cytochrome oxidase cannot be done in muscle because of Mb interferen,60 whereas it is possible in the brain cortex.61 Which Variable Should be Considered? Upon the premise that NIRS oxygenation signals reflect fractional O2 extraction (see below) in the investigated volume of tissue, the following question can be asked: is there a correlation between NIRS-derived oxygenation signals and PO2 or Hb saturation in venous blood draining from the exercising muscle(s)? The answer to this question may not be straightforward, and conflicting results have been reported in the past. The issue is complicated by the problem of obtaining, particularly in humans, adequate samples of venous blood coming directly from the exercising muscle(s), without a "contamination" from blood coming from other muscles and/or other tissues. Wilson et al.62 found an exllent correlation between a NIRS oxygenation index and O2 saturation determined in the venous blood draining from a dog gracilis muscle preparation. The data were subsequently confirmed in humans during forearm exercise.53 On the other hand, in two studies63,64 performed during constant work rate exercise on a cycle ergometer, the following phenomena were observed (in normoxia): a transient decrease in oxygenation following the transition to exercise, which was paralle by a decrease in femoral vein O2 saturation; these decreases were followed by a paradoxical muscle "reoxygenation," which was not associated with an increased O2 saturation in the femoral vein. The latter, as expected, remained low for the remaining portion of exercise. In other words, following exercise onset an association between the two variables (decreased muscle oxygenation, decreased femoral vein O2 saturation) was observed, whereas dissociation occurred during the remaining portion of the constant work rate exercise. A similar muscle oxygenation pattern has been observed in different muscles14,65,66 for the oxygenated-Hb and -Mb ([oxy(Hb+Mb)]) signal (see below) while not for [deoxy(Hb+Mb)], which increased and then stayed constantly elevated during the remaining portion of the exercise. September 2016 091313-5 Vol. 21(9) Grassi and Quaresima: Near-infrared spectroscopy and skeletal muscle oxidative function. . . Grassi et al.65 reasoned that the muscle reoxygenation suggested by the [oxy(Hb+Mb)] time-course could derive from an increased blood flow to the skin, occurring for thermoregulatory purposes, as also suggested by the results obtained by Maehara et al.,67 Chuang et al.,68 and Davis et al.69 An increased skin blood flow for heat dispersion would increase the [oxy(Hb+ Mb)] signal, whereas would not substantially affect [deoxy (Hb+Mb)], sin the enhand blood flow would not be associated with an increased gas exchange. This was substantially confirmed in a study carried out by Koga et al.37 These authors observed by continuous-wave NIRS (CW-NIRS) (see below and Ferrari et al.24 for a definition), during whole body heating, a more pronound increase of [oxy(Hb+Mb)] compared to that of [deoxy(Hb+Mb)]. The apparent increased oxygenation described by CW-NIRS was not observed when utilizing TDNIRS37 (see below). The confounding effects of an increased skin blood flow on oxygenation variables [in particular [oxy (Hb+Mb)] determined by CW-NIRS] have been confirmed in a study carried out by Messere and Roatta.70 These authors observed that most of the warming-indud increase of the sum between oxy- and deoxy-Hb and -Mb ([total(Hb+Mb)]) resulted from an increase in [oxy(Hb+Mb)], with a relatively small contribution by [deoxy(Hb+Mb)]. They also observed that the "contamination" of the muscle NIRS signal by an increased skin blood flow was substantially redud by using spatially resolved (SRS) NIRS (see below). No increases in muscle O2 saturation ( , see below) were observed, by utilizing SRS-NIRS, in the vastus lateralis of subjects performing dynamic knee-extension exercise at 20% of MVC after thigh heating at 37°C and 42°C.71 The unchanged was observed in the presen of a marked increase in cutaneous vascular conductan.54 Several studies have confirmed the presen of a good or very good correlation between the [deoxy(Hb+Mb)] signal, or other tissue oxygenation variables determined by NIRS, and venous O2 saturation, both in an animal model of isolated muscle in situ14,72,73 and in exercising humans.20,30,74,75 In Wüst et al.,72 the correlation was worse during the initial part of contraction period, which in that model is associated with a forful "muscle pump" effect and a substantial blood squeezing resulting from the sudden onset of tetanic contractions. The first NIRS instruments widely utilized on skeletal muscle adopted, as a deoxygenation index, the differential absorption of light between 760 and 800 (or 850) nm.52,53,67,76­83 The approach was based on the absorption characteristics of NIR light by the chromophores of interest at the two wavelengths. Subsequent instruments allowed the obtainment of relative (with respect to an initial value arbitrarily set equal to zero) or absolute [oxy(Hb+Mb)] and [deoxy(Hb+Mb)] values. The sum of [oxy(Hb+Mb)] and [deoxy(Hb+Mb)] indicates the total Hb + Mb volume ([total(Hb+Mb)]) in the tissue under consideration. Sin total [Mb] cannot change acutely during exercise, changes in [total(Hb+Mb)] would reflect a vasodilation or an increased capillary hematocrit in the tissue under consideration. Absolute values of the above-mentioned variables can be obtained by the more technologically sophisticated (and more expensive) SRS, TD, or frequency-domain (FD) instruments, whereas the less technologically sophisticated, less expensive, and more widely utilized CW instruments allow only relative values (for a more detai discussion on this topic, see below and Ferrari et al.18,21,24). This represents a limitation, which can be at least mitigated by performing a Journal of Biomedical Optics "physiological calibration" at the end of each test, by a transient ischemia of the investigated limb, obtained by applying for a few minutes a markedly suprasystolic pressure by a cuff, "upstream" of the region of investigation (see, e.g., Fig. 1 in Porlli et al.84). This maneuver is not too uncomfortable for the subject. The range of values of [deoxy(Hb+Mb)] obtained in the muscle between rest and the condition of full extraction (i.e., when the [deoxy(Hb+Mb)] signal reaches a "plateau" after 4 to 5 min of ischemia) should be related to the range of Cða-vÞO2m values between rest and full extraction. By adopting this physiological calibration, "semiquantitative" fractional O2 extraction values can be obtained, also by CW-NIRS instruments. Some controversy has been raised85,86 about which one of the two variables ([oxy(Hb+Mb)] or [deoxy(Hb+Mb)]) should more precisely reflect changes in skeletal muscle fractional O2 extraction. As discussed by Grassi,87 the time-course of [deoxy(Hb +Mb)] (while not that of [oxy(Hb+Mb)]) during a constant work rate exercise65 qualitatively reflects, rather closely, the time courses of variables related to fractional O2 extraction across a wide spectrum of experimental models, such as Cða-vÞO2 determined across a contracting isolated in situ muscle preparation88 or an exercising limb,11,20 microvascular PO2 (PO2mv ) determined by phosphoresn quenching in rat spinotrapezius muscle,89 intrallular PO2 determined by phosphoresn quenching in isolated amphibian muscle fiber.90 Similar kinetics between PO2mv and [deoxy(Hb+Mb)] during electrically stimulated contractions in the rat gastrocnemius were observed.91 A study described a very similar pattern, also, for deoxy-Mb determined by 1 H-MRS17. In other words, during constant work rate exercise (or isometric contractions of constant metabolic rate), a series of variables related to O2 extraction, spanning from Cða-vÞO2 across a whole limb to Mb saturation, show a very similar time-course: unchanged versus rest for a few seconds (suggesting an adequacy of _ _ QO2m with respect to VO2m --this information is relevant in _ terms of the factors determining the VO2 kinetics3,4,10), then _ a rapid exponential increase (the increase VO2m exeds the _ increase in QO2m ) to a new steady state, in which the ratio _ _ VO2m QO2m reaches a new equilibrium, although at a higher value compared to that at rest. In other words, [deoxy(Hb+Mb)] (while not [oxy(Hb+Mb)]) belongs to this family of variables related to fractional O2 extraction. This indirectly but strongly supports the role of [deoxy(Hb+Mb)] as an estimate of skeletal muscle fractional O2 extraction. The conpt is further strengthened by the issue of [oxy(Hb+Mb)] increases deriving from the enhand blood flow to the skin for thermoregulatory purposes, as discussed above. Strictly speaking, an increased [deoxy(Hb+Mb)] can be considered an estimate of an increased fractional O2 extraction only if [total(Hb+Mb)] is constant, which is not always the case in exercising skeletal muscles. However, there is ample eviden14,18,65,86,92­94 suggesting that changes in [deoxy(Hb+ Mb)] are less influend by changes in [total(Hb+Mb)] compared to changes in [oxy(Hb+Mb)]. Adami et al.92 observed a good correlation between the increase in [deoxy(Hb+Mb)] and the decrease in femoral blood flow during passive head_ up tilting, in the presen of a presumably unchanged VO2m . The data confirm the role of changes in [deoxy(Hb+Mb)] in evaluating O2 extraction in the presen of acute and passively _ indud changes in QO2m . In the study, passive head-up tilt indud also an increased venous blood volume, which September 2016 091313-6 Vol. 21(9) Grassi and Quaresima: Near-infrared spectroscopy and skeletal muscle oxidative function. . . complicated the interpretation of [deoxy(Hb+Mb)] as an index of O2 extraction;92 venous blood pooling is, however, unlikely to occur during exercise. In any case, the issue of the influen of blood volume has been substantially overcome by a rent study94 in which the authors calculated, on the basis of the assumption that during an arterial occlusion changes in [oxy (Hb+Mb)] and [deoxy(Hb+Mb)] should occur with a 11 ratio, a useful "blood volume correction factor," which can be applied to both [oxy(Hb+Mb)] and [deoxy(Hb+Mb)]. As mentioned above, SRS-, TD-, or FD-NIRS instruments allow the measurement of absolute values of [oxy(Hb+Mb)] and [deoxy(Hb+Mb)].24 By utilizing these values, a tissue oxygenation index (TOI) can be calculated, as the ratio [oxy (Hb+Mb)]/[total(Hb+Mb)].95 As an oxygenation variable, TOI is appealing sin it is expressed as a perntage, similarly to arterial O2 saturation (SO2a ). Sin it includes [oxy(Hb+ Mb)], however, also TOI may be more significantly influend by skin blood flow compared to [deoxy(Hb+Mb)]. Vogiatzis et al.30 utilized it to evaluate the intramuscular match_ _ ing between VO2m and QO2m . 3.4.2 Skeletal muscle O 2 consumption Physiological Variables of Functional Evaluation Which of the variables in the Fick equation [Eq. (1)] can be at least estimated by NIRS? The answer is: all of them, although in _ _ order to determine VO2m or Qm some specific (and rather unphysiological) or invasive protocols need to be adopted. When these protocols are not utilized, the oxygenation indis _ obtained by NIRS evaluate the dynamic balan between VO2m _ and QO2m . In other words, these indis allow the estimation of skeletal muscle fractional O2 extraction, a proxy of Cða-vÞO2m (see also below). 3.4.1 Skeletal muscle (or regional) blood flow De Blasi et al.96 determined forearm Q by NIRS, at rest and after hand exercise, by inducing a 50-mmHg venous occlusion by a cuff. They reasoned that if venous outflow from a muscle is impeded by the cuff, whereas arterial inflow is not (or at least is only partially impeded), the increase in [total(Hb _ +Mb)] as a function of time should be related to Q. The authors _ observed indeed an exllent correlation between forearm Q, determined by NIRS as described above, and the increase in forearm volume determined by plethysmography. A similar approach was adopted by van Beekvelt et al.32 No comparison _ with other methods to determine Qm was made. Limitations of the approach relate to the fact that pressure by the cuff inevitably limits, although only in part, also arterial inflow, as rently _ confirmed.97 Moreover, regional Q measurements could be carried out only during the recovery from exercise, and it is well known that the variable may rapidly change in the recovery phase.6 _ Qm in the exercising calf muscles has been determined in humans by utilizing NIRS in association with the intravenous infusion of the NIR trar indocyanine green (ICG).98 ICG is a fluoresnt dye approved by the U.S. Food and Drug Administration and European Medicines Agency for human use. Arterial blood was withdrawn by a pump and [ICG] was detected by photodensitometry, whereas NIRS optodes positioned over the muscle detected ICG transit in the microcircu_ lation at several wavelengths. Qm measurements were compared with those obtained by established methods. The main drawback of this elegant approach resides in its invasiveness, related to the need of obtaining arterial blood samples. The method has been _ utilized to determine Qm in respiratory99 and skeletal muscles100. Journal of Biomedical Optics Hamaoka et al.101 determined by NIRS the initial rate of Hb and Mb deoxygenation in finger flexors of humans during ischemia performed at rest and immediately after submaximal exercise of different intensities, and considered this variable a reflection of _ VO2m . These authors observed a significant correlation between this rate and the conntrations of intramuscular metabolites (PCr and adenosine diphosphate), determined by 31 P-MRS, considered to be regulators of oxidative phosphorylation. They concluded that NIRS can quantify the rate of oxidative metabolism at rest and after exercise. The validity of the approach was subsequently confirmed.102 The limitations are represented by the fact that the NIRS measurements need to be carried out in ischemic conditions and that the measurements are possible only in _ the recovery phase following exercise, during which VO2 rap6 idly decreases. While ischemic contractions do not represent a physiological condition, a "less unphysiological" experimental model would be one in which blood flow is held constant. Also in this case, according to the rearranged Fick equation [Eq. (2)], and after considering [deoxy(Hb+Mb)] a proxy of Cða-vÞO2m , the increase in [deoxy(Hb+Mb)] should represent an estimate of _ VO2m . This has indeed been experimentally confirmed in the study by Wüst et al.,72 which carried out in an isolated dog gas_ trocnemius in situ preparation. In the study, Qm was held con_ stant by a pump, VO2m was calculated by the Fick equation across the muscle and NIRS optodes were directly applied on the surfa of the exposed muscle. During metabolic transitions, the kinetics of adjustment of [deoxy(Hb+Mb)] was very similar _ to the kinetics of adjustment of VO2m . Brief sequential ischemic periods carried out during the recovery from exercise/contractions have been utilized in order to determine, by NIRS, the kinetics of recovery of _ VO2m following exercise.94,103­106 Also in this case, the rate of increase in [deoxy(Hb+Mb)] (and/or the rate of decrease in [oxy(Hb+Mb)]) during each of the brief ischemic periods _ is considered an estimate of VO2m . The kinetics of recovery _ of VO2m , as determined by the abovementioned protocol, has been shown to be closely correlated with two well-established variables of functional evaluation of skeletal muscle oxidative metabolism (frequently and incorrectly termed "oxidative capacity" by some of these authors): (1) the kinetics of recovery of [PCr], as determined by 31 P-MRS following exercise104 and (2) maximal adenosine diphosphate-stimulated mitochondrial respiration, as determined by high-resolution respirometry in isolated and permeabilized skeletal muscle fibers.106 3.4.3 Skeletal muscle fractional O 2 extraction As discussed above, in "normal" experimental conditions, oxygenation variables obtained by NIRS allow the evaluation of the _ _ dynamic balan between VO2m and QO2m , in other words of skeletal muscle fractional O2 extraction, a proxy of Cða-vÞO2m . NIRS oxygenation variables can represent only a proxy of Cða-vÞO2m sin they are usually expressed in relative terms, and the proportional contributions of arterial and venous blood to the overall signal are unknown. In the next paragraphs, we will provide examples of conditions in which analysis of September 2016 091313-7 Vol. 21(9) Grassi and Quaresima: Near-infrared spectroscopy and skeletal muscle oxidative function. . . skeletal muscle fractional O2 extraction appears relevant and of physiological interest. The capacity for prolonged exercise depends upon the capacity to supply the exercising muscles with O2 in order to satisfy the metabolic requirements. During exercise, however, fractional O2 extraction increases. In other words, compared to rest, the muscles (as other organs) utilize a greater perntage of the O2 they reive by the cardiovascular system. We will discuss, later in this section, how this increase in fractional O2 extraction could impair peripheral O2 diffusion and oxidative metabolism through the resulting decrease in microvascular PO2 (PO2mv ). Textbook physiology says that at the systemic level, in humans at rest, fractional O2 extraction is 25%, whereas during maximal exercise, the variable increases to 75%. In steady_ state conditions during exercise, the relationship between Q and _ _ VO2p is linear, with a slope of 5 (1 L min-1 increase in VO2p is _ accompanied by a 5 L min-1 increase in Q). Considering that 15 of the volume of arterial blood is represented by O2 (CaO2 200 mL L-1 of blood), the linear relationship between _ _ QO2 and VO2p has a slope of 1. As a consequen of the linear _ _ _ Q (or QO2 ) versus VO2p relationship, systemic Cða-vÞO2 would increase hyperbolically as a function of work rate. This has indeed been observed during ramp incremental cycling exercise.107 _ _ The relationship between Qm O2 and VO2m is somehow different when determined at the microvascular level, which is the domain investigated by NIRS. During an incremental or ramp exercise, the NIRS-derived [deoxy(Hb+Mb)] follows a sigmoid pattern,62,84,107­113 with a shallow increase at low work rates (sug_ _ gesting a tighter matching between muscle VO2m and QO2m , presumably associated with the recruitment of more oxidative fibers), a linear intermediate portion (suggesting greater O2 _ _ extraction and a progressively higher VO2m QO2m, presumably associated with the recruitment of less oxidative fibers), followed _ by a plateau at work rates approaching 80% of VO2m;max . At submaximal work rates, before the "plateau" is reached, the observation of higher fractional O2 extraction for the same _ VO2m (or work rate), in other words, a left-shifted [deoxy(Hb _ +Mb)] versus work rate curve would suggest an impaired QO2m . On the other hand, a lower fractional O2 extraction (right-shifted [deoxy(Hb+Mb)] versus work rate) would suggest an exss _ _ QO2m with respect to VO2m . This information may have profound physiological and pathophysiological significan (see below). The sigmoid shape of the [deoxy(Hb+Mb)] versus work rate curve has been shown to vary in muscles characterized by different fiber type compositions49 and has been confirmed (with minor differens) also during supine exercise114 as well as during incremental exercise in acute hypoxia.115 A right-shifted [deoxy(Hb+Mb)] versus work rate curve was described in trained cyclists.116 The [deoxy(Hb+Mb)] kinetics during constant work rate exercise has been utilized to evaluate the effects on the rate of adjustment of oxidative metabolism by a "prior" or "warm up" or "increased baseline" exercise,72,117­119 or to investigate the effects on microvascular fractional O2 extraction by interventions such as dietary nitrate supplementation, aimed at increasing microvascular nitric oxide (NO) availability.120 For example, the increased muscle oxygenation observed by Gurd et al.118 following a prior heavy intensity exercise suggests an enhand O2 availability, which should have contributed to _ the observed faster pulmonary VO2 kinetics. In other studies, the results were not conclusive. Bailey et al.,117 for example, Journal of Biomedical Optics observed after oral nitrate supplementation, a decreased muscle O2 extraction, which, however, could not be simply attributed to enhand O2 delivery sin it was associated with a decreased _ VO2 . Skeletal muscle fractional O2 extraction determined by NIRS can also give valuable physiological and pathophysiological insights into the integrated responses to exercise (and to other conditions) involving different muscle groups or skeletal muscles and other organs, as well as into the physiological _ mechanisms of Qm regulation. _ For example, arm Q (by thermodilution) and muscle oxygenation (by NIRS) were measured at the transition from _ arm to arm + leg exercise.66 During maximal exercise, arm Q _ decreased at the transition, to a degree that VO2m;max and muscle oxygenation were compromised. Thus, NIRS can give valuable _ _ insights into the coordination of Qm and VO2m adjustments to exercise between different muscle groups, in conditions of _ increased O2 demands and/or limited QO2m (see also below). Turner et al.121 observed [deoxy(Hb+Mb)] increases both in limb locomotor muscles and in respiratory muscles after interventions, such as inspiratory muscle loading performed during _ cycling exercise at 80% of VO2p;max , or after increasing work _ rate from 80% to 100% of VO2p;max . The results suggest a lim_ ited QO2m , both in locomotor and in respiratory muscles, which may impair exercise toleran during maximal exercise or in conditions of increased respiratory muscles work. By applying lower body negative pressure, Soller et al.122 indud a progressive decrease of ntral blood volume and a decreased stroke volume, which were paralle by a decreased muscle oxygenation from vasoconstriction. According to these authors, skeletal muscle deoxygenation determined by NIRS could be utilized as an early indicator of ntral hypovolemia and impending cardiovascular collapse. NIRS has also been utilized to elucidate basic physiological _ mechanisms of Qm regulation, such as the metabolic modulation of sympathetic vasoconstriction (functional sympatholysis) by exercise123 or by tissue hypoxia.124 By simultaneously measuring sympathetic nerve activity by microelectrodes and forearm oxygenation by NIRS, Hansen et al.123 provided eviden in favor of functional sympatholysis. Reflex sympathetic activation, which consistently decreased oxygenation in resting forearm muscle, had no effect on oxygenation when the muscles were exercised. The [deoxy(Hb+Mb)] plateau during an incremental exercise can be considered an estimate of the maximal capacity of O2 extraction during exercise and has been utilized in several studies84,109­113,125­127 as a variable of functional evaluation of skeletal muscle oxidative metabolism. Venous blood draining from exercising muscles still contains small but significant amounts of O2 ; in other words, fractional O2 extraction is not 100%.128­130 Which are the normal values of fractional O2 extraction across skeletal muscle during maximal exercise? According to Knight et al.,128 during cycling, the value can go up to 90%, as determined by invasive measurements carried out across an exercising limb. A fractional O2 extraction value of 85%, also in this case determined invasively across an exercising limb, was observed during maximal knee-extensor exercise,130 which is an exercise paradigm (involvement of a relatively low small muscle mass) in which cardiovascular constraints to O2 delivery are not present or are significantly redud, and skeletal muscle blood flow can be three times higher than during conventional cycle ergometer exercise. September 2016 091313-8 Vol. 21(9) Grassi and Quaresima: Near-infrared spectroscopy and skeletal muscle oxidative function. . . These values are very similar to those obtained in normal subjects by NIRS for [deoxy(Hb+Mb)] at peak exercise, expressed as a perntage of the maximal [deoxy(Hb+Mb)] value observed during a transient limb ischemia.84 _ Although, in normal subjects, VO2p;max is traditionally con_ sidered to be mainly limited by maximal cardiac output (Qmax ) _ and maximal cardiovascular O2 delivery (QO2 max ),131 the role of peripheral factors cannot be overlooked.132,133 If one considers the Fick equation [see Eq. (1)] applied to conditions of maximal exercise, it is obvious that the main differen between subjects _ _ with high VO2p;max and subjects with low VO2p;max resides in _ 2 max , and not in Cða-vÞO2 max . However, the conpt that QO _ QO2 max represents cardiovascular limitations and Cða-vÞO2 max or maximal fractional O2 extraction represent peripheral factors is an oversimplification.134 In an in-series O2 transport system, every step must play a role in affecting the overall outcome, and _ the different functions are strictly interconnected.133 QO2 , by setting microvascular PO2 (PO2mv ), determines peripheral O2 diffusion and therefore fractional O2 extraction. In the presen _ of an elevated QO2 , as it occurs in athletes or after training, peripheral O2 diffusing capacity (DO2m ) must increase in order to allow a large fractional O2 extraction. Although the issue is debated,135 in skeletal muscle, an O2 _ extraction limit is present, which contributes to VO2p;max and _ VO2m;max limitation; moreover, a limitation in peripheral O2 diffusion appears to be the major factor responsible for the limited extraction.133 Exercise training may increase muscle fractional O2 extraction by elevating muscle(s) DO2m to a greater extent _ than QO2m;max .35,42 Kalliokowski et al.136 observed by positron emission tomography that fractional O2 extraction during exercise is higher in enduran trained athletes compared to untrained controls. According to Whipp and Ward,137 systemic _ Cða-vÞO2 max increases hyperbolically with VO2p;max . In other words, also in healthy subjects, fractional O2 extraction is rel_ evant in determining VO2p;max . This conpt applies even more strongly to patients or to conditions in which oxidative metabolism is impaired (see below). The "snario" depicted above can be looked at also from another perspective: an increased O2 extraction may impair peripheral O2 diffusion. This could occur also during submaximal exercise. A fall in oxygenation (corresponding to an _ _ increased VO2m QO2m ) suggests a less pronound or inad_ equate vascular response in relation to an increased VO2m . This would determine a decreased PO2mv and mean capillary PO2 , a decreased driving pressure for peripheral O2 diffusion from capillaries to mitochondria,42,43,138 and a functional impairment of skeletal muscle oxidative metabolism. The phenomenon could also occur transiently in the early phase of a constant work rate exercise. This is suggested by the transient "overshoot" of skeletal muscle fractional O2 extraction (usually determined by analyzing the [deoxy(Hb+Mb)] time-course), which has been described in contracting in situ dog muscle14 as well as during constant work rate exercise in different populations (see also below), such as patients with chronic heart failure (CHF),95,139 patients with metabolic myopathies,140 type 2 diabetes,141 chronic obstructive pulmonary disease,142 healthy subjects undergoing bed rest deconditioning84 or bed rest associated with hypoxia,127 healthy subjects during arm exercise.81 In the latter study, the overshoot in skeletal muscle fractional O2 extraction, determined by NIRS, was closely associated with an overshoot in Cða-vÞO2 , determined invasively across the exercising muscles. Goodwin Journal of Biomedical Optics _ et al.14 experimentally slowed QO2m kinetics in isolated in situ dog muscle and observed an enhand [deoxy(Hb+Mb)] over_ shoot and a slowed VO2m kinetics. In human subjects acutely exposed: to normobaric hypoxia, Bowen et al.33 observed a slower VO2p kinetics and a more pronound [deoxy(Hb+Mb)] transitory increase, although the magnitude of the overshoot was unaffected. The O2 extraction overshoot, occurring early during a constant work rate exercise, is considered to be caused by a _ _ transient mismatch between QO2m and VO2m .42,43,138,143,144 The mismatch could derive from a suboptimal NO signaling within the muscle, which could "uncouple," both temporally and spa_ tially, the heterogeneous microvascular QO2m increase from the _ 2m increase.43,138 presumably heterogeneous VO The transiently increased fractional O2 extraction is associated with a transiently decreased PO2mv .42,43,138,144 This phenomenon has been described in animal models, in which PO2mv was determined by phosphoresn quenching.35,145 As mentioned above, the decreased PO2mv would redu the driving pressure for peripheral O2 diffusion and impair oxidative metabolism. The impairment would be functionally significant also because it would be temporally located in the critical early phase following an increase in metabolic intensity.3,4,6,10 A partially different approach to the [deoxy(Hb+Mb)] overshoot has been taken by Murias et al.146 These authors utilized _ a "normalized" ½deoxyðHb þ MbÞVO2p ratio, in which both variables are expressed as a perntage of the response. In _ order to calculate the ratio, the VO2p signal is left-shifted by 20 s, which is assumed to represent, on average, the delay _ between the VO2m increase (the variable of interest in the com_ parison with [deoxy(Hb+Mb)]) and the VO2p increase,3,4 which is the variable usually determined. Another assumption related _ _ to this approach is that VO2p kinetics equals VO2m kinetics. This is not exactly true (see the discussion in Koga et al.42): the two variables have similar kinetics11 but with a substantial intersub_ ject variability. Moreover, [deoxy(Hb+Mb)] and VO2p increases are, in relative terms, substantially different, and therefore, the normalization (perntages of responses) likely introdus a distortion in the calculation of the ratio. In another approach, Ferreira et al.143 assumed (as Murias _ _ et al.146 did) that VO2p kinetics equals VO2m kinetics, and that [deoxy(Hb+Mb)], as an estimate of O2 extraction, quantitatively reflects Cða-vÞO2 ; the authors then calculated microvascular blood flow adjustment by solving the Fick equation. The main limitations of this approach, in our opinion, reside in the _ _ assumptions: the first one (VO2m kinetics equals to VO2p kinetics) has been discussed above, but also the second one ([deoxy(Hb+Mb)] as a quantitative measurement of Cða-vÞO2 ) is not strictly correct: even when convincing "physiological calibration" methods are utilized, [deoxy(Hb+Mb)] allows at best only a semiquantitative estimation of Cða-vÞO2 , and therefore, its use in replacing Cða-vÞO2 in mathematical equations seems questionable.147 Moreover, the approach by Ferreira et al.143 assumes that the contribution by Mb to the NIRS signals is zero; as discussed above, this is likely not the case. _ During the recovery following exercise, the decrease in VO2p _ and VO2m follows an exponential time course, even in the presen of a sudden decrease in mechanical power output.6 _ In these conditions, the "exss VO2m ," with respect to the needs of myosin ATPase, derives essentially from the resynthesis of PCr, which occurs by oxidative phosphorylation. Upon the assumption of equilibrium of the CK reaction, the recovery rate of [PCr] following exercise is therefore a function of September 2016 091313-9 Vol. 21(9) Grassi and Quaresima: Near-infrared spectroscopy and skeletal muscle oxidative function. . . mitochondrial ATP production. The rate of recovery of [PCr] following exercise has often been utilized as a tool of evaluation of skeletal muscle oxidative metabolism.15,104 Another variable frequently utilized as a functional evaluation tool is the NIRSdetermined reoxygenation kinetics during the recovery. This kinetics is a function of the interplay between the kinetics of _ _ QO2m and the kinetics of VO2m , and cannot discriminate between the two. However, a good agreement was found between the rate of recovery of [PCr] (as determined by 31 P-MRS) and the reoxygenation kinetics (as determined by NIRS) following moderate-intensity exercise that is the absen of significant drops in pH.80 These authors concluded that the reoxygenation kinetics during the recovery from a moderateintensity exercise could represent a functional evaluation tool of oxidative performan. 3.4.4 Other variables and purposes Several studies have been conducted with the aim to identify, by NIRS, variables of functional evaluation of oxidative metabolism (thresholds), such as the ventilatory threshold,148,149 the "EMG threshold,"150 the onset of blood lactate accumulation during incremental exercise,151 or the "maximal lactate steady state."152 Most of these studies observed a ni correlation between NIRS-obtained indis and more traditional variables utilized to identify the various thresholds.148,149,151,152 Broxterman et al.153 utilized NIRS, together with EMG and the analysis of the curvature constant of the power versus time to exhaustion relationship, in order to evaluate the role of ntral and peripheral fatigue during exercise with small muscle mass (handgrip) in conditions of blood flow occlusion. ntral (brain) factors were identified as responsible for fatigue during submaximal elbow flexors exercise in severe hypoxia;154 in the study, the authors utilized muscle and brain NIRS, evoked and EMG responses to electrical muscle stimulation, transcranial magnetic stimulation, and motor-evoked potentials. Soller et al.122 developed a mathematical model, which correlated NIRS spectra with muscle pH (pHm ) values obtained invasively by sensors plad in the muscle. They could then predict pHm values on the basis of NIRS spectra, and determined, during incremental exercise, an "Hþ threshold," which correlated with classic "metabolic thresholds". Towse et al.155 utilized NIRS blood volume and muscle oxygenation changes to confirm the blood-oxygenation-leveldependent (BOLD) mechanism underlying the changes of magnetic resonan imaging signal intensity observed following brief, single contractions. Several NIRS studies have been performed in electrically stimulated contractions. For instan, McNeil et al.156 observed by NIRS a greater metabolic demand, associated with greater fatigue during electrically stimulated (versus voluntary), contractions carried out at the same torque production. On the contrary, the local O2 demand of the bips brachii was found similar between electrical muscle stimulation and maximal voluntary isometric contractions.157 versus young activity-matched controls, in association with _ _ higher Qm and QO2m and an unchanged microvascular fractional O2 extraction. These results point to a higher energy cost of contractions as the main factor responsible for the redud exercise capacity in the elderly. At least in part, different results were described by Ferri et al.,125 who observed in 78-year old subjects, higher skeletal muscle (vastus lateralis) fractional O2 extraction, at the same absolute work rate, suggesting an _ impairment of QO2m . This phenomenon was observed both during cycle ergometer exercise (in which the limitations to oxidative metabolism are mainly related to cardiovascular O2 delivery) and during one-leg knee-extension exercise,125 in which cardiovascular limitations are substantially redud.130 _ VO2p kinetics is slower in the elderly. Murias et al.159 observed that this slower kinetics was associated with a larger _ overshoot in the ½deoxyðHb þ MbÞVO2p (both variables were expressed as a perntage of their total response, see also above) ratio, suggesting, according to the authors, an impaired adjust_ ment of QO2m . This would be in agreement with observations of a blunted vasodilatory dynamics in arterioles of old rats.160 _ Exercise training speeded VO2p kinetics and redud the _ 2p overshoot,159 suggesting an improved ½deoxyðHb þ MbÞVO _ _ matching between QO2m and VO2m . In another study, no _ ½deoxyðHb þ MbÞVO2p overshoot was observed in chronically trained elderly subjects.161 It should be considered, however, that in these studies,159,161 the overshoot in [deoxy(Hb _ +Mb)] was only relative to VO2 ; in other words, no "real" [deoxy(Hb+Mb)] overshoot was present. Immobilization/Microgravity and Countermeasures Higher skeletal muscle fractional O2 extraction for the same work rate, a redud peak capacity of O2 extraction during an incremental exercise, and an overshoot of [deoxy(Hb+Mb)] during constant work rate exercise were observed in healthy young subjects undergoing 35 days of bed rest.84,111 These findings were observed during both cycle ergometer exercise84 and one-leg knee extension exercise.111 These results include skeletal muscle oxidative metabolism among the "victims" of profound inactivity. Sin bed rest studies have been considered a "proxy" of microgravity (see the discussion in Porlli et al.,84 Salvadego et al.111), the same conpt applies to microgravity. Superposition of normobaric hypoxia (equivalent to an altitude of about 4000 m) upon 10 days of bed rest attenuated, during one-leg knee extension exercise (but not during cycle ergometer exercise), the impairment of the peak capacity of fractional O2 extraction and the magnitude of the overshoot, which were observed following bed rest alone.127 An enhand perfusive and diffusive O2 delivery deriving from the hypoxia-indud increased [Hb] may explain, at least in part, this unexpected finding. Training programs carried out during 21-day forearm immobilization by a plaster cast were found effective in preventing the decline in muscle oxidative function, enduran, and strength.162 Aging, Immobilization, Hypoxia Aging Hypoxia By combining 31 P-MRS, Doppler ultrasound imaging, and NIRS Layec et al.158 observed a higher ATP cost of contractions during plantar flexion exercise in elderly subjects (73-year old) Journal of Biomedical Optics During acute moderate normobaric hypoxia (inspired O2 partial pressure [PO2I ] 118 mmHg, equivalent to an altitude of 2500 m), muscle deoxygenation during an incremental cycle exercise is not significantly different, for the same absolute work rate, compared to normoxic conditions.163 The data September 2016 091313-10 Vol. 21(9) Grassi and Quaresima: Near-infrared spectroscopy and skeletal muscle oxidative function. . . _ suggest higher Qm in hypoxia compared to normoxia and, thus, the existen of control mechanisms, which effectively match _ _ QO2m to VO2 . This effective matching is presumably lost at higher altitudes. Billaut et al.164 collected muscle and brain oxygenation data to gain insights into the ntral and peripheral factors affecting muscle fatigue in conditions of limited O2 delivery. They described a trend toward lower vastus lateralis muscle oxygenation in subjects performing repeated sprints and acutely exposed to a slightly more pronound (compared to that of the previously mentioned study) moderate hypoxia (FO2I ¼ 0.138, PO2I 98.4 mmHg). Subudhi et al.165 described greater fractional O2 extraction values for the same absolute work rate in subjects acutely exposed to hypobaric hypoxia (PO2I 86 mmHg) corresponding to an altitude of 4300 m. Interestingly, no differens versus acute hypoxia were described, in the same subjects, in chronic hypoxia (5-day acclimatization at 1830 m and 1-day at 4300 m). In all the mentioned studies,163­165 marked differens between normoxia and acute or chronic hypoxia were described for prefrontal cortex oxygenation. Unchanged muscle oxygenation during exercise was described after versus before a 72-day acclimatization to chronic hypobaric hypoxia (Mt. Everest expedition);166 the unchanged muscle deoxygenation was associated with an increased hypoxic toleran and with enhand acute hypoxic ventilatory response and rebral oxygenation. In another Mt. Everest expedition, a slower muscle (thenar eminen) reoxygenation kinetics was observed during vascular occlusion tests carried out at 4900 and 5600 m.167 A more pronound muscle deoxygenation during constant work rate exercise was described during acute hypoxia,67 obtained either by having the subjects breath air with a FO2I of 0.15 or by carbon monoxide loading aimed at increasing carboxyhemoglobin saturation. Interestingly, the aclerated deoxygenation was described only during exercise above the "lactate threshold," that is, at an exercise intensity at which O2 supply to the exercising muscles becomes critical.2,3,4,10 A greater deoxygenation of acssory respiratory muscles during voluntary isocapnic hyperpnea was observed in hypoxia (FO2I ¼ 0.10 - 0.11) versus normoxia,168 in association with a higher muscle activation determined by EMG. Patients Populations In this section, we will present an overview of some representative studies in which the variables of functional evaluation discussed in the previous section have been determined in adult patients' populations. The aims were to evaluate the evolution of impairments as a function of time, to identify correlations with clinical symptoms, to evaluate the effects of therapeutic or rehabilitative interventions, and to gain pathophysiological and diagnostic insights. A systematic review on muscle NIRS measurements in the clinical care of term and preterm neonates has been rently published.169 Chronic Heart Failure Several studies have been conducted by utilizing NIRS in patients with CHF. The complex interplay between ntral and peripheral factors in determining the pathophysiology of the disease and the impaired exercise toleran in CHF patients has been discussed in detail in the review by Poole et al.138 A subsequent review by the same group144 discussed, from Journal of Biomedical Optics the same perspective, the effects of training in this patient population. Wilson et al.62 observed higher fractional O2 extraction, for the same absolute work rate, in CHF patients versus agematched healthy controls; the results suggest an impaired _ QO2m in the patients, presumably a consequen of the impaired cardiac function. A similar pattern was observed by another study in CHF patients.126 Interestingly, in this study, the _ impaired QO2m response was not affected by 3 months of light-to-moderate exercise training. The training intervention, on the other hand, increased the maximal capacity of skeletal muscle fractional O2 extraction during exercise, allowing the patients to reach values not different from those of controls. A very similar pattern to that described in CHF patients126 before training was observed in patients evaluated after a sucssful heart transplantation,109 carried out in the treatment of severe CHF. The data suggest that the surgical intervention, although improving cardiac function, did not significantly _ improve the QO2m impairment at submaximal exercise and the redud capacity of maximal fractional O2 extraction by skeletal muscles. Also in patients undergoing heart transplantation, a complex interaction between ntral and peripheral functions limits exercise toleran.109 Sperandio et al.139 described by NIRS in CHF patients, during constant work rate submaximal exercise, a transient overshoot in fractional O2 extraction (increased [deoxy(Hb+Mb)]), presumably leading to a decreased PO2mv and to an impaired peripheral O2 diffusion during the critical early phase of the metabolic transition. This overshoot could derive from a suboptimal intramuscular NO signaling.138 The [deoxy(Hb+Mb)] overshoot is qualitatively equivalent and has the same pathophysiological meaning with respect to the transient PO2mv undershoot described in the spinotrapezius muscle of CHF rats.170 In addition, Sperandio et al.139 observed that in CHF patients an increased NO bioavailability, obtained by the administration of sildenafil, eliminated the [deoxy(Hb+Mb)] overshoot, determined a faster _ VO2p kinetics, and improved exercise toleran. Bowen et al.95 observed that the deoxygenation overshoot in CHF patients was attenuated by a prior exercise (aimed at enhancing _ _ QO2m ), which determined also a faster VO2p kinetics, confirming the link between the overshoot and an impaired oxidative metabo_ lism. On the basis of the deoxygenation and VO2p kinetics, the authors identified two subgroups of patients: the most severely limited ones were those with an impaired skeletal muscle oxidative metabolism.95 Belardinelli et al.82 observed in CHF patients a slower reoxygenation kinetics following exercise, in association with a _ slower VO2p kinetics. In another study, Hanada et al.171 observed that the slower reoxygenation kinetics in CHF patients was associated with a markedly slower kinetics of recovery of [PCr], as determined by 31 P-MRS. As mentioned above, a slower reoxygenation kinetics during the recovery after exercise is considered an index of impaired skeletal muscle oxidative performan.80 At least in part, different results were obtained by Kemp et al.:172 according to these authors, in CHF patients the slowing of muscle reoxygenation kinetics was more pronound than the slowing of [PCr] recovery; the data suggest _ a QO2m limitation. _ A slower recovery kinetics of VO2m (determined by NIRS by utilizing the method by Ryan et al.94 mentioned above) following wrist-flexor exercise was observed in systolic CHF patients;173 interestingly, whereas in control subjects, a 4September 2016 091313-11 Vol. 21(9) Grassi and Quaresima: Near-infrared spectroscopy and skeletal muscle oxidative function. . . week period of exercise training determined a faster recovery kinetics, this did not occur in the CHF patients. The authors concluded that the impairment of nonlocomotor muscles oxidative metabolism in the CHF patients was not reversible with a short period of exercise training. Moreover, the impairment was not simply due to deconditioning, but suggests a skeletal muscle dysfunction intrinsically associated with the disease, as also proposed in the reviews by Poole et al.138 and Hirai et al.144 Mancini et al.76 observed deoxygenation of acssory muscles of respiration (serratus anterior) in CHF patients (but not in controls) during submaximal and maximal cycle exercise; the observed deoxygenation may contribute to the exertional dyspnea typically described in these patients. It is well known that more severely affected CHF patients typically have a hyperventilatory response to exercise, which holds clinical and prognostic implications.174 The hyperventilatory response, the decreased respiratory muscle strength, and the _ increased work of breathing would increase QO2m requirements of respiratory muscles, and could indu a "competition" for the _ limited available QO2 between respiratory and locomotor muscles, contributing to the decreased exercise toleran.175 In support of this hypothesis, Borghi-Silva et al.176 observed that respiratory muscles unloading, obtained in CHF patients by proportional assisted ventilation, increased exercise toleran in association with an enhand leg muscle oxygenation and blood volume determined by NIRS. These studies demonstrate how NIRS can give valuable pathophysiological insights into the interplay between different muscles in conditions of limited _ available QO2 . A functional evaluation of skeletal muscle oxidative metabolism would be even more important in CHF patients with preserved ejection fraction, in whom peripheral impairments appear more significant than those usually observed in the more "traditional" CHF patients with redud ejection fraction.144,177 In this respect, very few data are available yet. Peripheral Arterial Disease and Diabetes During incremental exercise, PAD patients show near-maximal deoxygenation (with respect to the ischemic calibration) beginning from very low work rates.79 In the same study, the reoxygenation kinetics following exercise was markedly slower in PAD patients; moreover, the half-time of this recovery was significantly correlated with the "ankle-arm index" (ratio of systolic blood pressures at the ankle and at the arm),79 a clinical variable evaluating the severity of the disease. A markedly slower reoxygenation kinetics in PAD patients was confirmed in subsequent studies.83,141 In PAD patients, the involvement of the two legs is often markedly asymmetrical. Interestingly, McCully et al.79 observed that the reoxygenation kinetics was markedly slower in the "bad" leg versus the "good" leg. According to McCully et al.,83 analysis of reoxygenation kinetics by NIRS could be utilized as a screening tool for the presen of PAD. In patients with PAD, NIRS has also been utilized to evaluate the effects of various therapeutic interventions. Whereas Beckitt et al.178 described only modest improvements of muscle oxygenation during exercise in PAD patients treated with angioplasty, substantial positive effects were observed after the administration of sildenafil179 or after an oral supplementation (beetroot jui) of nitrates.180 Both these approaches aimed, although by exploiting different pathways, at increasing NO bioavailability in the microvasculature, thereby taking advantage of Journal of Biomedical Optics the vasodilatory effect of NO and also at improving the intra_ _ muscular matching between VO2m and QO2m . What is the pathophysiological meaning of the slower reoxygenation kinetics in PAD patients? As a general rule, this kinetics is a function of the interplay between the kinetics of _ _ QO2m and the kinetics of VO2m during the recovery following exercise. In PAD patients, the first variable is obviously impaired, but the second one could be impaired as well. This has been demonstrated by Bauer et al.,141 who observed in PVD patients a slower recovery of reoxygenation also in the patients with reasonably normal calf blood flow responses, as evaluated by plethysmography. The clinical relevan of NIRS applications in PAD has been very rently reviewed by Boezeman et al.,181 who considered 183 manuscripts. These authors found sufficient eviden to use NIRS in the clinical setting for assessment of chronic compartment syndrome of lower extremities, and as a surveillan tool for the detection of "free flap failure". On the other hand, the clinical relevan of routine use of NIRS in other vascular diseases was found to be less clear. _ For the same absolute work rate and VO2p , type 1 diabetic patients show (versus controls) higher [deoxy(Hb+Mb)], sug_ _ gesting an impaired QO2m , as also suggested by the lower Qm.182 An overshoot of [deoxy(Hb+Mb)] during constant work rate exercise has been described in patients with type 2 diabetes.183 _ This observation was associated with slower VO2p kinetics and _ appears compatible with an impaired QO2m adjustment, consequen of the microvascular impairment. The [deoxy(Hb+Mb)] overshoot described in diabetic patients is qualitatively equivalent and has the same pathophysiological meaning of the transient PO2mv undershoot described in the exercising muscles of diabetic rats.89 In a review, Pedersen et al.184 discussed the main methods to evaluate mitochondrial dysfunction in patients with PAD and type 2 diabetes. According to these authors, NIRS works better than 31 P-MRS in determining the severity of the impairment. Manfredini et al.185 utilized NIRS in order to find objective correlates between the onset of symptoms of claudication and the limited exercise toleran in PAD patients, with and without diabetes. In order to do so, they determined the area under the [oxy(Hb+Mb)] or [deoxy(Hb+Mb)] curves (with respect to the baseline) during an incremental exercise. These areas evaluate (although in arbitrary units) the overall burden represented by _ _ the dynamic unbalan between VO2m and QO2m . The areas were greater in the PAD patients with diabetes and were correlated with the onset of symptoms. In the PAD patients with diabetes, however, ischemic symptoms were delayed and occurred at a level of deoxygenation which was significantly greater than in the patients with PAD alone, possibly as a consequen of a blunted pain perption and/or of a subjective higher toleran for muscle discomfort associated with diabetes. Chronic Obstructive Pulmonary Disease By substituting nitrogen with helium in inspired air, gas density is redud to 13 of that of normal ambient air. After relieving the work of breathing by having COPD patients breathe a normoxic helium-O2 mixture, Chiappa et al.186 observed less leg _ deoxygenation by NIRS, faster VO2p kinetics, and an increased exercise toleran. The authors hypothesize that by decreasing _ the competition for the available QO2 between respiratory and locomotor muscles, helium-O2 breathing could increase leg _ QO2m , thereby enhancing skeletal muscle oxidative metabolism. September 2016 091313-12 Vol. 21(9) Grassi and Quaresima: Near-infrared spectroscopy and skeletal muscle oxidative function. . . This hypothesis was only in part confirmed in a subsequent study.48 These authors determined by NIRS, by utilizing the ICG dye dilution method,98 intercostal, and quadrips muscles _ Qm in COPD patients breathing room air or a normoxic helium_ O2 mixture. During cycling exercise at 75% of VO2p;max , _ helium-O2 was associated with increases in Qm in both intercostal and quadrips muscles, together with an increased CaO2 and with signs of enhand exercise toleran. The authors hypothesized that changes in lung mechanics indud by : helium-O2 likely increased stroke volume, Q and systemic _ QO2 . A subsequent study187 went a step further. These authors subdivided COPD patients into dynamic "hyperinflators" and _ "nonhyperinflators," measured quadrips Qm by the ICG _ method98 and calculated QO2m . Helium-O2 increased exercise _ toleran and QO2m in both groups, but at least in part different mechanisms were involved: in hyperinflators, helium-O2 _ increased CaO2 and QO2m in the presen of: no changes in : Q; in nonhyperinflators, helium-O2 improved Q, vascular con_ ductan, and QO2m . These studies nily demonstrate that, also in COPD patients, NIRS can give valuable insights into the mechanisms regulating the integrated responses to conditions of increased O2 demand. Also, COPD patients showed the [deoxy(Hb+Mb)] overshoot during constant work rate exercise142 and a slower reoxygenation kinetics during the recovery following exercise.188,189 Following 6 weeks of high-intensity training, the reoxygenation kinetics became faster, in association with an increased citrate synthase activity and an increased exercise toleran.188 Metabolic Myopathies and Muscular Dystrophies Sin NIRS oxygenation indis reflect skeletal muscle fractional O2 extraction, they appear to be ideally suited to identify and evaluate the impairment of oxidative metabolism in patients with mitochondrial myopathies (MM) and myophosphorylase deficiency (McArdle disease, McA), in whom the incapacity to increase skeletal muscle fractional O2 extraction represents a key pathophysiological mechanism (see the discussion in Grassi et al.110). Following the pioneering work by Bank and Chan,78 Grassi et al.,110 using NIRS, measured [deoxy(Hb+ Mb)] at exhaustion (½deoxyðHb þ MbÞpeak ) during an incremental exercise test to obtain the maximal capacity of skeletal muscle fractional O2 extraction in MM and McA patients. The values were significantly lower than those of controls, and they _ were linearly correlated with VO2p;peak ,110 a close approximation _ of VO2p;max ; they also were inversely related to the time-constant _ of VO2p kinetics,190 another classical variable of functional evaluation of skeletal muscle oxidative metabolism.3,4,10 The most severely impaired patients could not increase their fractional O2 extraction during exercise compared to the values obtained at rest, confirming observations obtained in previous studies by invasive measurements (see the discussion in Grassi et al.110). Moreover, ½deoxyðHb þ MbÞpeak values obtained in MM and McA patients were substantially lower than those obtained in patients with similar signs and symptoms but with a negative biopsy for any known metabolic myopathy.110 In other words, NIRS allowed one to identify and quantify the metabolic impairment in MM and McA patients and could also offer diagnostic clues, for example, in the selection of patients on whom to perform a muscle biopsy. Markedly lower [deoxy(Hb+Mb)] values at submaximal work rates were observed in MM and McA patients.110,140 Journal of Biomedical Optics Lower [deoxy(Hb+Mb)] values at the same submaximal work rate, versus controls, were also observed in MM during incremental handgrip exercise.191 These data suggest an exss in _ _ QO2m for the same VO2m (and work rate), which represents one of the key pathophysiological mechanisms of the diseases. MM and McA patients also showed a marked transient [deoxy(Hb+Mb)] overshoot during constant work rate exercise.140 In McA, the overshoot disappeared in the presen of the "second wind" phenomenon, a sudden decrease in heart rate and perived exertion occurring in these patients after a few minutes of exercise, or during a second exercise preded by a few minutes by a prior exercise.140 The second wind phenomenon is thought to derive from an enhand sympathoadrenal response, which would allow the utilization of glucose deriving from blood or extramuscular sours and of free fatty acids, thereby bypassing the metabolic impairment (the inability to breakdown intramuscular glycogen) at the basis of the disease (see the discussion in Porlli et al.140). In other words, in McA patients, NIRS confirmed the mechanism (sudden enhanment of oxidative metabolism) at the basis of the second wind phenomenon. Another glycolytic defect leading to a decreased capacity to increase fractional O2 extraction during exercise is muscle-type phosphofructokinase (M-PFK) deficiency. The impairment has been demonstrated by NIRS in humans70 and in dogs.192 ½deoxyðHb þ MbÞpeak was also determined by Marzorati et al.193 in a small population of patients with the adult form of Pompe disease (glycogen storage disease type II, due to the deficiency of the lysosomal enzyme acid maltase), before and after 12 months of enzyme replament therapy. Following enzyme replament therapy, the authors observed that ½deoxyðHb þ MbÞpeak slightly increased in some of the patients. In patients with Friedreich's ataxia, the genetic defect decreases the expression of the protein frataxin, localized in mitochondria. The resulting impairment of mitochondrial function and oxidative metabolism has been identified at the skeletal muscle level by 31 P-MRS (see the discussion in Lynch194). These authors observed by NIRS a slower reoxygenation kinetics following exercise, and the degree of impairment of this variable correlated with the length of the triplet GAA repeat, a genetic measure associated with the age of onset of the disease.194 Quaresima and Ferrari195 first investigated by NIRS quadrips muscle oxygenation in patients with Duchenne muscular dystrophy (DMD). An insufficient increase in _ _ QO2m with respect to VO2m occurs in these patients.196 In other words, the functional defect is the opposite of that characterizing MM and McA patients (see above). The dystrophin deficiency results in a loss of sarcolemmal neuronal NO. NO plays a key role in the control of muscle blood flow during exercise, by blunting the vasoconstrictor response to -adrenergic stimulation.123,124 In DMD patients, the impaired NO synthesis would impair the intramuscular matching of _ _ QO2m versus VO2m , leading to an increased O2 extraction, which has indeed been demonstrated by NIRS.196 An enhand O2 extraction has been observed197 also in patients with Becker muscular dystrophy, a milder form of dystrophy in which the intramuscular NO impairment is also present. In these patients, the enhand O2 extraction was associated with the functional impairment. An enhand O2 extraction, on the other hand, has not been described in patients with September 2016 091313-13 Vol. 21(9) Grassi and Quaresima: Near-infrared spectroscopy and skeletal muscle oxidative function. . . limb-girdle muscular dystrophy, in whom neuronal NO synthase is not impaired.196 Caliandro et al.198 could differentiate, by utilizing arterial occlusion testing in postocclusion hyperemia, patients with dermatomyositis and inclusion-body myositis from healthy controls; the differentiation was not possible for patients with polymyositis. A mitochondrial dysfunction, evaluated by the _ recovery kinetics of [PCr] (by 31 P-MRS) and VO2m (by NIRS), was observed in patients with amyotrophic lateral sclerosis.199 Other Diseases Other patients populations characterized by a skeletal muscle oxidative impairment, which was identified by NIRS by the _ analysis of the rate of decrease in VO2m following voluntary or electrically stimulated contractions,94,200 are those with cystic fibrosis201 or spinal cord injuries.202 In a case report, Ryan et al.200 described a threefold increase of the rate of decrease _ in VO2m (indicating a substantial improvement of skeletal muscle oxidative metabolism) in a patient with a spinal cord injury who underwent 12 weeks of enduran training, conducted by neuromuscular electrical stimulation. Malenfant et al.203 observed by NIRS, in patients with pulmonary arterial hypertension, markedly higher [deoxy(Hb+ Mb)] during submaximal exercise at the same absolute work rate versus controls, indicating a markedly higher vastus lateralis fractional O2 extraction. The data suggest an impaired _ QO2m in these patients, which would be compatible with the lower capillary density observed in the same muscle of the other leg. The data would also be compatible with the increases in endothelin-1 levels and with the decreases in NO and prostaglandins. Interestingly, hyperoxic breathing, while normalizing SO2a , did not substantially affect the [deoxy(Hb+Mb)] _ response, confirming an impairment in QO2m deriving from the structural and functional impairments mentioned above. In patients with fibromyalgia, Shang et al.204 combined the utilization of a commercially available NIR oximeter with a custom-built NIR diffuse correlation spectroscopy (DCS) instru_ ment for tissue Qm measurements. Experiments were conducted during fatiguing knee extensors isometric contractions and a forearm arterial occlusion protocol. From the O2 extraction _ _ and Qm data, VO2m was calculated (see also below). The patients showed, versus the controls, a lower fractional O2 extraction and a slower reoxygenation kinetics. Muscle NIRS measurement is an important tool in identifying high-risk patients in septic and hemorrhagic shock, by offering a continuous and noninvasive monitoring of tissue hypoperfusion.205,206 According to these authors, during active shock resuscitation changes in skeletal muscle oxygenation levels are correlated with changes in O2 delivery, base deficit, and blood lactate levels. A drop in muscle oxygenation levels predicts death and multiorgan failure, and may be helpful in making critical clinical decisions. Instrumentation Advanments The present section is aimed at briefly reporting the advanments made in the last few years in the field of NIRS instrumentations suitable for muscle studies and at mentioning the future directions of the development/utilization of these NIRS devis. The main commercial NIRS oximeters for muscle studies, with the related key features and measurable parameters, are listed in Table 1. Sin 2011 (when a homologous table was Journal of Biomedical Optics published in the review by Ferrari et al.24), different batteryoperated portable/wireless muscle oximeters have been commercialized. The CW-NIRS portable/wearable systems, with wireless data transmission, are reported in Table 2. The details about each system can be found in the related company's websites. Three different NIRS techniques are utilized in the field of muscle oximetry. Each of them is based on a specific type of illumination: (1) the most commonly used CW instruments, based on constant illumination of the tissue, simply measure the attenuation of light through the tissues; (2) FD instruments, illuminating the tissues with intensity-modulated light, measure both the attenuation and the phase delay of the emerging light; and (3) TD instruments, illuminating the tissue with short pulses of light (few picoseconds), detect the temporal broadening of the pulse after propagation through the tissues. The quantitation of the NIRS measurable parameters depends on the adopted NIRS technology. Different NIRS methods using the CW modality have been developed to measure muscle O2 saturation ( , %). The measurement assures an accurate quantitation of the oxygenation changes occurring at the skeletal muscle level. The CW-based systems offer the advantages of low-cost and easy transportability. In asnding order, CW-, FD-, and TD-based instruments require increased cost and technological complexity. On the other hand, FD and TD techniques offer a better depth discrimination than that achieved by CW instruments and allow the absolute characterization of the tissue optical properties (absorption and redud scattering coefficients), from which it is possible to retrieve absolute conntration values of [oxy(Hb+ Mb)] and [deoxy(Hb+Mb)], and the derived parameter [total(Hb+Mb)]. The available CW-NIRS instruments do not determine redud scattering coefficient and circumvent the problem by assuming that the coefficient is substantially constant during rest and exercise. This assumption has been criticized.207 The issue has also been discussed in the review by Ferrari et al.24 Most of the NIRS instruments reported in Table 1 are represented by two to six channels and CW brain oximeters utilized also for physiological/pathophysiological muscle studies. These oximeters are the most commonly utilized NIRS instruments because they are of relatively low/moderate cost and transportable. Muscle NIRS instrumentations are characterized by a relatively high temporal resolution; the sampling rate is up to 100 Hz in some CW systems, 50 Hz in the only commercial FD system, and 0.2 Hz in the first TD oximeter with mark, commercialized in 2015 (Table 1). An original muscle oximeter has been very rently introdud by Reflectan Medical; the instrument collects muscle spectra and calculates as well as pHm . The sector of wearable health technology is gaining increasing interest. The use of low cost wearable devis or wearable biosensors, which allow a constant monitoring of physiological signals, is essential for the advanment of both the diagnosis and the treatment of diseases, as well as for monitoring active life style. Very rently, a textile NIRS system has been utilized for monitoring arterial O2 saturation (measured by pulse oximetry) and muscle oxygenation changes.208 Wearable wireless fingertip pulse oximetry devis, for arterial O2 saturation measurement, are already available for smart phones. In addition, a wireless and smartphone controllable NIRS system (DynaSense September 2016 091313-14 Vol. 21(9) Grassi and Quaresima: Near-infrared spectroscopy and skeletal muscle oxidative function. . . Inc., Japan) has been very rently presented.209 Table 2 includes also two wireless systems that are waterproof, then allowing muscle oxygenation measurements in swimmers.210 For a better understanding of skeletal muscle physiology, muscle oxygenation data should be ideally integrated with regional blood flow measurements. Direct and indirect NIRS _ methods to measure Qm 20,96 have been discussed above. A novel technique, known as DCS, has been rently developed for quantifying relative changes in microvascular regional blood flow. Basically, DCS uses coherent NIR light to penetrate deep tissues and measures speckle fluctuations of the diffuse light that are sensitive to the motions of red blood lls in tissues.211,212 The well validated DCS provides a portable, noninvasive, and inexpensive alternative for measuring microvascular blood flow. A rent review article has described the _ progresses made in the field of DCS for assessing Qm during 213 dynamic exercise. This technology allows also the estimation of skeletal muscle oxygenation levels. So far, two DCS-NIRS systems have already been commercially available: FloMo (Hemophotonics S.L., Spain) and MetaOx (ISS, _ United States). The former measures only Qm, while the latest provides concomitant measurements of and absolute [oxy (Hb+Mb)] and [deoxy(Hb+Mb)] by FD-NIRS. The method has been utilized on the head of newborns and on the skeletal muscles of adults. Giovannella et al.214 have integrated the -FloMo with a TD-NIRS prototype (TRS20, Hamamatsu _ Photonics, Japan) to measure Qm and absolute [total(Hb+ Mb)] during quadrips exercise. A different approach for meas_ uring Qm has been utilized by Ornim Inc. (United States), which has developed the brain/muscle oximeter "rOx 3210F" using NIRS and weak acoustic beams to identify light emerging from deep tissue layers. Many sophisticated multichannel CW-NIRS instrumentations, dedicated to brain cortex two-dimensional (2-D) imaging (topography), are commercially available and largely utilized in different areas in the field of neuroscien.25,215 Unfortunately, the probes they are equipped with cannot be utilized for skeletal muscle 2-D imaging studies. In 2015, an eight-measurement point wireless system for 2-D brain/muscle measurements has been commercialized (Table 2). In the framework of a National Aeronautics and Spa Administration (NASA) project, a 64-measurement point wireless NIRS system has been developed and integrated with simultaneous electrocardiography and actigraphy monitoring, in order to record 2-D muscle imaging data up to several days.216 Although a threedimensional (3-D) NIR imaging (tomography) of the human forearm, based on TD techniques, had been proposed217 almost 15 years ago, no further consistent progresses in this field have been made. Rently, cross-sectional 2-D images of the foot [total(Hb+Mb)], using a multichannel 3-D CW system during thigh cuff occlusion, were reported.218 The state-of-the-art of TD-NIRS has been rently reviewed.219 Many progresses have been done in the last 2 years that open a new way to compact, low-cost, and highperforman TD devis for diffuse muscle optical imaging and spectroscopy. The new frontiers in TD diffuse optics are covered by an article of this Special Section. 220 Briefly, consistent progresses are expected conrning noncontact scanning 2-D imaging systems for TD-NIRS based on a null sour­ detector separation.221­223 These systems, already tested in the human forearm, utilize new fast detectors for extreme depth penetration and sensitivity.224,225 Journal of Biomedical Optics Conclusions Researchers should always be critical and careful about the technologies and methods they utilize, but within reasonable limits. Many exercise physiologists, in our opinion, have been exssively critical about NIRS in the past. The method has important limitations but presents significant advantages compared to other methods. Skeletal muscle fractional O2 extraction, the main variable that can be noninvasively evaluated by NIRS, can yield relevant information on key physiological and pathophysiological mechanisms, relevant in the evaluation of exercise performan and exercise toleran in healthy subjects and in patients. To put the method in a broader perspective it should be recognized, for example, that the BOLD phenomenon,226 as obtained by functional magnetic resonan imaging, a functional neuroimaging technique which has profoundly revolutionized neuroscien with applications ranging from normal brain development, aging, brain disorders and diseases, is conptually very similar to the fractional O2 extraction variable determined by NIRS in skeletal muscle. Both variables, indeed, substantially reflect changes in tissue oxygenation resulting from the dynamic balan between tissue O2 consumption and O2 supply. The rent diffusion of apparently easy-to-use portable NIRS instruments had to the proliferation of studies, sometimes of dubious quality. An example is represented by studies, which claim to obtain absolute oxygenation indis when the instrumentation they employ does not allow this. The authors must be aware of the limitations of the method and should always have a clear and sound hypothesis to test. However, in the right hands (a good physiological background is mandatory), NIRS can offer insights into key physiological and pathophysiological adaptations to conditions of increased O2 needs, often involving, in an integrated manner, different organs of the body. In terms of patients' evaluation, NIRS allows to determine the evolution of functional impairments of skeletal muscle oxidative metabolism with time, to identify correlations with clinical symptoms, to evaluate the effects of therapeutic or rehabilitative interventions, and to gain pathophysiological and diagnostic insights. 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Chen, "Metabolic and vascular origins of the BOLD effect: implications for imaging pathology and resting-state brain function," J. Magn. Reson. Imaging 42, 231­246 (2015). Bruno Grassi, reived his MD and PhD degrees and now is a professor of physiology in the Medical School of the University of Udine (Italy), where he is head of the Exercise Physiology Laboratory. His research interests are in the fields of exercise and environmental physiology, skeletal muscle bioenergetics, with special referen to oxidative metabolism, in health and disease. He is the author of 112 scientific publications on international peer-reviewed journals and of 25 chapters in books. Valentina Quaresima reived her PhD and now is an associate professor of biochemistry at L'Aquila University, Italy. She has pioneered clinical applications of NpIRS of the adult and infant muscle/brain and has given substantial contributions to wider NIRS scientific community. Sin 1993, she has been involved in the study of localized measurements of muscular and rebral oxygenation in humans by different NIRS techniques. She is the author of 100 scientific publications on international peer-reviewed journals. Journal of Biomedical Optics 091313-20 September 2016 Vol. 21(9)

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Journal of Biomedical OpticsSPIE

Published: Sep 1, 2016

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