The Use of Skeletal Muscle Near Infrared Spectroscopy and a Vascular Occlusion Test at High Altitude

Introduction

With increasing altitude, the partial pressure of atmospheric oxygen declines as a consequence of the reduction in atmospheric pressure. Humans possess the capacity to adapt to this hypobaric hypoxia through the process of acclimatization, classically characterized by the augmentation of systemic convective oxygen transport mediated through increases in minute ventilation, heart rate, and hemoglobin concentration (Hornbein and Schoene, 2001; West et al., 2007). In addition, innate physiological alterations within the tissue microcirculation might help match local tissue oxygen demands with supply. However, pathophysiological microcirculatory disregulation could uncouple the oxygen supply-demand relationship, leading to tissue hypoxia and cellular metabolic failure (Ince, 2005). Such influences have yet to be characterized and clarified: a reduction in microcirculatory flow appears to be a dominant feature in both animal models (Saldivar et al., 2003) and human volunteers ascending to altitude (Martin et al., 2009a, 2010), although whether this represents a beneficial or detrimental adaptation remains unclear.

Skeletal muscle oxygenation can be measured by near infrared spectroscopy (NIRS) and, in combination with a noninvasive vascular occlusion test (VOT), provides a dynamic assessment of resting oxygen consumption and microvascular reactivity (Gerovasili et al., 2010). We hypothesized that adaptation to hypobaric hypoxia at high altitude (acclimatization) would result in i) reduced skeletal muscle oxygen consumption, and ii) impairment of the microcirculation, as assessed (respectively) by the decline in muscle oxygenation during a brief period of ischemia, and its rate of recovery upon cessation of the ischemic challenge.

Methods

Measurement of thenar eminence muscle oxygen saturation

Local oxygenation in skeletal muscle can be monitored by NIRS, which calculates tissue oxygen saturation (Sto₂) from the ratio of oxygenated to total hemoglobin beneath a light-emitting optode. The absolute concentrations of oxy- and deoxyhemoglobin cannot be calculated by NIRS unless the path-length of the light through the tissue of interest is known (Delpy and Cope, 1997). NIRS also detects other chromophores in blood and tissue, and myoglobin and other species can comprise up to 80% of the overall optical signal in human skeletal muscle (Marcinek et al., 2007). However, the deoxgenation signal in skeletal muscle is primarily (≈80%) derived from hemoglobin (Mancini et al., 1994). Static Sto₂ values therefore vary widely between individuals, whereas dynamic physiological challenges evoke changes in Sto₂, the rate of which reliably reflects changes in tissue oxygen tension. The NIRS-VOT consists of a short period of skeletal muscle ischemia induced by an occlusive cuff proximal to the site of NIRS probe, and has been used to assess skeletal muscle microcirculatory function in both healthy volunteers and patients ( Bezemer et al., 2009; Creteur et al., 2007; De Blasi et al., 1993, 2005). The procedure typically involves placing a blood pressure cuff around the upper limb, rapidly inflating it above systolic blood pressure for a period of 3–5 min, and then releasing the cuff. During the protocol, Sto₂ is measured continuously by NIRS, distal to the cuff, and the measurements plotted to generate a Sto₂-time curve (Fig. 1). Temporary arterial, and venous, occlusion throughout cuff inflation results in muscle ischemia, and a steady decline in Sto₂. This reaches a nadir prior to cuff release, which is followed by rapid reperfusion, re-oxygenation, and a reactive hyperemia (marked by a period of Sto₂ raised above baseline). There is then a slow return of Sto₂ back to baseline. The decline in muscle Sto₂ during occlusion (Sto₂ downslope) has been used as a measure of skeletal muscle oxygen consumption (Cheatle et al., 1991; De Blasi et al., 1993), whilst the rate of increase in Sto₂ after cuff release (Sto₂ upslope) is facilitated by a well-functioning microcirculation (Neviere et al., 1996).

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FIG. 1.

A typical Sto₂-time curve generated during a near-infrared spectroscopy vascular occlusion test. AUC, Area under curve.

Skeletal muscle in the thenar eminence was selected for the site of NIRS-VOT in this study as the thenar eminence is minimally susceptible to the confounding effects of excessive adipose accumulation and tissue edema (Gerovasili et al., 2010). Sto₂ was measured using a tissue spectrometer (InSpectra Model 325, Hutchinson Technology, MN, USA), fitted with a probe of 15 mm optode spacing, and was defined as the ratio of oxygenated to total haemoglobin concentration. The device used light at 680 nm, 720 nm, 760 nm, and 800 nm and measured Sto₂ every 3 sec. Data were recorded onto a laptop computer for later analysis.

Subjects rested for a minimum of 10 min in a sitting position prior to the commencement of recordings. Their dominant arm was placed at the height of the heart and a manual blood pressure cuff place around the upper arm. A NIRS probe was attached to the thenar eminence and taped around its edges to reduce any effect that excessive ambient light might produce. Subjects were advised that they must not move their arm during any part of the investigation. After a 3 min period of stabilization, baseline Sto₂ recordings were commenced. The blood pressure cuff was rapidly inflated to 250 mmHg for 3 min, then deflated. NIRS data collection continued for 3 min after cuff release. The exact times of occlusion and deflation were electronically marked on the NIRS tracing.

Results

At sea level the mean (±SD) Spo₂ was 97.9 (±0.6)%, which was significantly reduced at 4900 m, 83.6 (±5.4)%, and 5600 m, 77.6 (±7.1)%, (p<0.001). The mean temperature in the laboratories was 24.1 (±1.0) ^oC at sea level, 20.7 (±2.9) ^oC at 4900 m, and 17.6 (±3.4) ^oC at 5600 m. Individual Sto₂-time curves were generated for all participants at each of the three altitudes (Figs. 2 to 4) and analyzed using the Inspectra software. The mean values for all NIRS-VOT measurements are shown in Table 2.Mean baseline Sto₂ was 84.4 (±6.0)% at sea level and declined to 69.3 (±8.2)% at 4900 m (p=0.001) and 64.2 (±6.1)% at 5600 m (p<0.001). The downslope during arterial occlusion remained unchanged between sea level and high altitude (Fig. 5) but the upslope, post cuff release, was significantly reduced at 4900 m (2.4 (±0.4)%/sec) and 5600 m (2.4 (±0.8) %/sec) when compared to sea level, (3.7 (±1.3)%/sec) (p=0.021 and p=0.032, respectively) (Fig. 6). There was no difference between upslope measurements at 4900 m and 5600 m (p=1.00). There was also no difference between genders in this subject cohort for any of the NIRS-VOT measurements. The Inspectra software was not able to calculate area under the hyperaemia curve reliably in more than 30% of NIRS-VOT traces due to insufficient post-ischemic data. Analysis of the area under the hyperemic curve was therefore not performed.

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FIG. 2.

Individual Sto₂-time curves for subjects during a near-infrared spectroscopy vascular occlusion test at sea level. Arterial occlusion from 0 to 180 seconds.

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FIG. 3.

Individual Sto₂-time curves for subjects during a near-infrared spectroscopy vascular occlusion test at 4900 m above sea level. Arterial occlusion from 0 to 180 seconds.

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FIG. 4.

Individual Sto₂-time curves for subjects during a near-infrared spectroscopy vascular occlusion test at 5600 m above sea level. Arterial occlusion from 0 to 180 seconds.

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FIG. 5.

Sto₂ downslope measurements for subjects at sea level, 4900 m, and 5600 m. 4900 m=Day 7 at altitude; 5600 m=Day 17 at altitude.

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FIG. 6.

Sto₂ upslope measurements for subjects at sea level, 4900 m, and 5600 m. 4900 m=Day 7 at altitude; 5600 m=Day 17 at altitude; NS=nonsignificant.

At both of the high altitudes (4900 m and 5600 m), there was a significant inverse correlation between minimum Sto₂ during the NIRS-VOT and subsequent Sto₂ upslope; r²=0.348 and p=0.044 at 4900 m, and r²=0.608 and p=0.003 at 5600 m. This relationship was absent at sea level (r²=0.065 and p=0.424). When data from both high altitudes (4900 m and 5600 m) were combined, there were weak but statistically significant correlations between Spo₂ and baseline Sto₂ (r²=0.206, p=0.03) and Spo₂ and peak recovery Sto₂ (r²=0.523 and p<0.001). There was no correlation between Spo₂ and either Sto₂ minimum, downslope or upslope. However, there was also a correlation between the Sto₂ downslope and Sto₂ upslope in the combined altitude cohort (r²=0.555 and p<0.001). This relationship was not present at sea level.

Discussion

This is the first reported use of NIRS with a VOT at high altitude, here applied to the thenar eminence. Ascent to altitude was associated with systemic arterial hypoxemia and also with tissue hypoxia (mean thenar eminence Sto₂ being lower at rest, and during and at peak after 3 min of complete arterial occlusion). Ascent also appeared associated with unaltered basal oxygen consumption (Sto₂ downslope during occlusion) despite the presence of tissue hypoxia, but with impaired microcirculatory function (reduced Sto₂ upslope, Fig. 6)—Sto₂ upslope reflecting endothelium dependent vasodilation (Gerovasili et al., 2010).

That systemic hypoxemia was associated with a sustained reduction of resting baseline skeletal muscle Sto₂ is, perhaps, to be expected: skeletal muscle hypoxia is demonstrable in the presence of hypoxemia (Ferrari et al., 1997; Subudhi et al., 2007), and thigh muscle Sto₂ correlates with the oxygen saturation of its venous outflow when healthy volunteers breathe hypoxic gas mixtures (Costes et al., 1996). More importantly, however, we showed altitude exposure to be associated with a sustained reduction in Sto₂ upslope. This impact seemed independent of the absolute degree of arterial hypoxemia: despite Spo₂ falling from 83.6 % at 4900 m to 77.6 % at 5600 m (p<0.001), there was no further reduction in absolute Sto₂ measurements or the upslope (2.4%/sec at both altitudes). We also demonstrated weak correlation between Spo₂ and two of the absolute Sto₂ measurements (baseline and peak recovery) at altitude, and between Sto₂ up and downslopes at altitude.

The divorce between the degree of arterial hypoxemia and the magnitude of depression in Sto₂ upslope is hard to explain with certainty. However, the responses we characterized will have resulted from a complex interaction of degree and magnitude of hypoxic exposure along with an unpredictable degree of acclimatization. Whilst the magnitude of hypoxia was greater at 5600 m, there was a period of 10 days between the two high altitude measurements. Absence of a further decline in Sto₂ upslope may be explained by adaptive changes within skeletal muscle occurring over this time interval. Interactions between exposure-duration and exposure-magnitude are difficult to unravel in the setting of a field study, however repeated recordings over time at each altitude might have provided additional insights into the mechanisms underlying these findings. Alternatively, the decrease in Spo₂ from 4900 m to 5600 m may have been insufficient to elicit additional alterations within the skeletal muscle microcirculation, or the upslope may have reached its minimum physiological rate.

Sto₂ upslope represents removal of deoxyhemoglobin from the ischemic skeletal muscle by a rapid influx of oxyhemoglobin when arterial influx returns (Bezemer et al., 2009). However, NIRS-assessed StO₂ upslope is not a measure of blood flow per se, but a surrogate for a spectrum of endothelially-dependent changes in capillary blood flow (Poole et al., 2011) which occur following a period of ischemia. Vasodilatation begins during the ischemic occlusion phase such that when the cuff is released, there is a brief period of hyperemia, the magnitude of which is dependent of microcirculatory function (Neviere et al., 1996). Endothelial dysfunction thus results in slower reperfusion rates and therefore a reduced Sto₂ upslope (Gerovasili et al., 2010). We have previously demonstrated that ascent to high altitude is associated with a reduction in sublingual microcirculatory flow index (MFI) measured by sidestream dark field (SDF) imaging (Martin et al., 2009a, 2010). However, it remains unclear as to whether this reduction in MFI and alteration of Sto₂ upslope represents a pathological or beneficial adaptive response to hypoxemia. It is plausible that a reduced microcirculatory blood flow may aid tissue oxygenation through an increase in tissue transit time.

However, other factors may also have contributed to the observed changes in Sto₂ upslope. Tissue diffusion limitation, the potential barrier to oxygen flux from erythrocytes to cells when arterial oxygenation falls, and the gradient of oxygen tension between them is diminished (Wagner, 1992), may have played such a role. In addition, metabolic mediators (such as adenosine, potassium, hydrogen ions, carbon dioxide, and prostaglandins) certainly play a central role in mediating exercise-induced hyperemia, (Delp and Laughlin, 1998) and may also influence the hyperemic response to ischemia. Thus, the vasodilatory effect of hypoxemia could have exhausted skeletal muscle flow reserve prior to the ischemic stimulus commencing, giving the impression of a diminished vascular response after the VOT. Increased sympathetic/ catecholaminergic activity at altitude (Cunningham et al., 1965), detectable in skeletal muscle ( Mazzeo et al., 1995; Saito et al., 1988), may also have affected the microcirculation and thus Sto₂ upslope. Polycythemia (Huff et al., 1951), endothelial dysfunction (Vallet, 1998), and changes in red blood cell deformability (Parthasarathi and Lipowsky, 1999) following acclimatization may also have impacted upon the microvascular flow response. Low ambient temperature could (through vasoconstriction) have affected Sto₂ upslope, although the reduction in ambient temperature in the high altitude laboratories was not particularly marked.

The relationship between Sto₂ up- and downslope also warrants further comment. Whilst these measures were unrelated at sea level, they correlated at altitude such that a reduced resting muscle oxygen consumption was associated with a less responsive microcirculation. This might represent evidence of the coupling of metabolic and circulatory responses. Perhaps present at sea level but only revealed in response to sustained hypoxia. The lack of change in Sto₂ downslope in the current study suggests that there was no overall change in resting muscle oxygen consumption at altitude.

The NIRS-VOT technique has been validated (Kragelj et al., 2000). Whilst data vary considerably between individuals (Figs. 2 to 4) (likely due to differences in subcutaneous adipose tissue depth and the inability of the Hutchinson spectrometer to generate a truly quantitative measurement), intra-individual variability remains low. In the present study, 3 minutes of arterial occlusion allowed adequate time for useful readings to be measured without causing undue discomfort, and its use has been reliably applied and reported by others investigators (Gerovasili et al., 2010). Active limb movement during measurements will increase muscle oxygen consumption. The need for passive immobility was, however, stressed to all subjects, and any unseen movement is unlikely to have introduced systematic bias.

The literature is divided as to whether cuff inflation should be for a specific time period (Creteur et al., 2007) or to a pre-defined minimum Sto₂ (Gomez et al., 2008). We selected the time-limited methodology so as to have a body of clinical data to draw comparison with and to avoid confounding from the inter-individual differences in absolute Sto₂ that occur on ascent to altitude (Martin et al., 2009b).

The Inspectra NIRS device measured Sto₂ at 3 second intervals. The analysis software generated Sto₂ curves by extrapolating between measurement points and the accuracy of data relating to periods of rapid change (such as VOT recovery) may thus have been blunted. Furthermore, the NIRS–VOT measurements were calculated by commercially written software that contained predetermined algorithms. Such factors are unlikely to introduce any altitude-related bias, but may limit comparison with studies using different analysis techniques. These technical differences might account for lower baseline Sto₂ upslope values in our study when compared to those reported by Creteur et al. ( 2007) and Doerschug et al. (2007) (3.7%/sec vs. 4.8%/sec and 4.7%/sec, respectively). The inability of the Inspectra software to calculate the hyperemic area under the curve in all cases was due to insufficient measurement time post cuff release. In retrospect, a longer period of data capture post cuff release may have avoided this data loss.

Some degree of tissue edema is not uncommon upon ascent to altitude (Gunga et al., 1995; Maggiorini et al., 1990). If present within the skeletal muscle or overlying subcutaneous tissue, this might have attenuated the NIR light signal and falsely lowered recorded Sto₂ values. However, altitude-related accumulation of interstitial edema at the thenar eminence (our site of measurement) is uncommon (Mancini et al., 1994; Poeze, 2006).

We observed no difference between genders in the NIRS–VOT measurements studied, however, imbalance in the gender distribution amongst the subjects (four females, eight males) may account for this negative finding, and a type 2 error cannot be excluded.

Conclusion

Ascent to high altitude resulted in hypoxemia that was associated with a reduction in skeletal muscle absolute Sto₂ values. Basal oxygen consumption, as assessed by Sto₂ downslope during vascular occlusion, was unaffected by hypoxemia at altitude. However, the recovery rate after occlusion had been released (St -o₂ upslope) was significantly reduced. These observations may reflect alterations in microvascular reactivity, but whether these are physiological or pathophysiological remains unknown. The results of this study may have implications for critically ill patients exposed to sustained hypoxemia secondary to pathophysiology.