Methodological considerations for near-infrared spectroscopy to assess mitochondrial capacity after spinal cord injury

Abstract

Background: Skeletal muscle mitochondrial activity is reduced by ∼ 50–60% after SCI, resulting in impaired energy expenditure, glucose utilization and insulin sensitivity. Near infra-red spectroscopy (NIRS) is a non-invasive tool that can be used to assess mitochondrial capacity.

Objectives: (1) Highlight methodological limitations impacting data acquisition and analysis such as subcutaneous adipose tissue (SAT) thickness, movement artifacts, inadequate muscle stimulation, light interference, and ischemic discomfort. (2) Provide technical considerations to improve data acquisition and analysis. This may serve as guidance to other researchers and clinicians using NIRS.

Study Design: cross-sectional observational design.

Settings: Clinical research medical center.

Participants: Sixteen men with 1 > year post motor complete SCI.

Methods: NIRS signals were obtained from right vastus lateralis muscle utilizing a portable system. Signals were fit to a mono-exponential curve.

Outcome Measures: Rate constant and r² values for the fit curve, indirectly measures mitochondrial capacity.

Results: Only four participants produced data with accepted rate constants of 0.002–0.013 s⁻¹ and r² of 0.71–0.87. Applications of studentized residuals ≥2.5 resulted in sparing data from another four participants with rate constants of 0.010–0.018 s⁻¹and r² values ranging from 0.86–0.99.

Conclusions: Several limitations may challenge the use of NIRS to assess mitochondrial capacity after SCI. Acknowledging these limitations and applying additional data processing techniques may overcome the discussed limitations and facilitate data sparing.

Introduction

Mitochondria play a pivotal role in cellular energy production through oxidative phosphorylation, which utilizes electrochemical gradients and oxygen molecules, as a final acceptor, to pump electrons across mitochondrial membranes; this yields water and over 95% of the energy (adenosine triphosphate [ATP]) body requirements.^1,2 Mitochondrial capacity is determined by the maximal affinity of utilizing oxygen and can be used as a surrogate to assess the overall health in different clinical populations. Mitochondrial disfunction can decrease energy production, increase reactive oxygen species released, alter cellular redox status, or even promote cellular apoptosis.^3,4 This has been observed in aging and neurological disorders such as traumatic brain injury and spinal cord injury (SCI).^5,6 Mitochondrial dysfunction has also been observed in individuals with type II diabetes, obesity, and SCI by almost 50%–60%.^5–8

In the U.S., approximately 18,000 new SCI cases are reported annually, with a prevalence of more than 280,000.⁹ The lifetime cost for an individual with SCI may exceed $4.8 million.⁹ Due to decreased mobility, physical activity impairment, and poor dietary habits, SCI is often accompanied by deterioration in body composition and metabolic health.^10,11 This is characterized by rapid onset of skeletal muscle atrophy, infiltration of intramuscular fat (IMF), increase in visceral adipose tissue, and reduction of basal metabolic rate.¹⁰ These changes have been linked to increased risk of obesity, type II diabetes, and cardiovascular disease.^10–13 Additionally, recent work has indicated that mitochondrial density and activity are tightly correlated with both body composition and metabolic profile in persons with motor complete SCI.¹⁴ The authors suggested that mitochondrial dysfunction may precede several of the previously described changes. These findings seem to indicate that mitochondrial dysfunction may precede skeletal muscle atrophy through cascades of protein ubiquitination and degradation.¹⁵ Furthermore, the restoration of lean and muscle mass may improve mitochondrial density and activity after SCI.¹⁶

Historically, mitochondrial capacity has been assessed in vitro utilizing muscle tissue biopsies^17,18, or through in situ phosphorous magnetic resonance spectroscopy (MRS).¹⁹ However, muscle biopsies are invasive and both methods are expensive, requiring a high degree of technical expertise while still not capturing in vivo conditions.^17–20 Considering the significance of studying mitochondrial capacity after SCI, there is an essential need for an alternative, non-invasive technique.

A noninvasive, cost-effective, and relatively simple in vivo technique to assess mitochondrial capacity utilizes near-infrared spectroscopy (NIRS) combined with a series of arterial occlusions.^21,22 NIRS technology relies on wavelength-dependent absorption of near-infrared light by hemoglobin and myoglobin molecules as it propagates through different tissues to measure tissue oxygenation.^23,24 Brief muscular activity (10–15 s) during an arterial occlusion eliminates free ATP and phosphocreatine (PCR) stores within a target muscle. This is followed by the gradual recovery of oxygen supplies and replenishment of ATP and PCR substrates. The rate of oxygen recovery provides an indirect measure of mitochondrial capacity for the tested muscle. This technique appears to provide a robust to assessment of mitochondrial capacity as it correlates with the rate of oxygen consumption in skeletal muscle fibers;^21,22 furthermore, it has been cross-validated with muscle biopsy²² and MRS.²⁵ These techniques have also been utilized in other clinical populations including SCI.^26,27

Based on the potential of the NIRS serial arterial occlusion technique, we initiated a clinical study aimed at using this non-invasive tool to assess skeletal muscle mitochondrial capacity in persons with SCI. However, several methodological and/or technical limitations hindered the processes of data acquisition, processing, and analysis. The purpose of the current work is to highlight and expand on these limitations and provide some considerations that may enhance applications of NIRS in assessing skeletal muscle mitochondrial capacity after SCI.

Methods and materials

Participants

Participants were recruited from a convenience pool as a part of an ongoing parent clinical trial registered at clinicaltrials.gov (NCT01652040). All participants were males with chronic (≥ 1-year post SCI), motor complete or incomplete (American Spinal Injury Classification: A, B), aged between 18 and 65 years with a body mass index (BMI) < 30 Kg/m². All study procedures were approved by a local ethics committee and each participant provided written consent prior to testing. Following consent, participants underwent a physical examination by a board-certified physician to confirm eligibility.

Testing preparation and device setup

Upon arrival, participants were instructed to void their bladder. Resting heart rate and blood pressure were then assessed (Vital Signs Monitor 6000 Series, Welch Allyn, Inc.) before participants were transferred from their wheelchair to a mat using a ceiling lift. Participants were laid in a supine position for at least 10 min, during that time the skin was prepared by removing excess hair and cleaning with alcohol pads. A pillow was provided for head and neck support and additional bolsters were provided to stabilize the knee joint.

The amount of oxygenated (O2Hb), deoxygenated hemoglobin (HHb), and total hemoglobin (THb) were measured using a portable NIRS unit (PortaMon, Artinis Medical Systems, Elst, Netherlands). The PortaMon was selected because it was easy to use and cost-effective;²⁸ additionally, the PortaMon comes with proprietary software to assist with data acquisition. The NIRS device was positioned longitudinally on the belly of the vastus lateralis muscle of the right thigh ∼10 cm above the patella and secured with medical tape. A vascular cuff (Hokanson SC-10D, Hokanson, Inc., Bellevue, WA) was placed as proximally as anatomically possible on the right thigh and controlled by a rapid-inflation system (Hokanson E20, Hokanson) set to maintain a pressure of 250 mmHg. To exercise the target muscle, surface neuromuscular electrical stimulation (NMES; Thera touch 4.7; Richmar, Inola, OK) was applied during testing via two 8 × 10 cm² surface electrodes (Uni-Patch, Wabasha, MI). One electrode was placed laterally ∼2–3 cm above the superior aspect of the patella, and the other was seated on the proximal thigh, lateral to, and ∼30 cm above the patella. A biphasic waveform pattern was administered with frequency (5 Hz), current intensity (175 mA), and pulse- duration of 450 µs. Figure 1 shows representative equipment setup on a participant’s thigh.

Figure 1.

The appropriate placement of the NIRS system, NMES electrodes, and arterial cuff over the target vastus lateralis muscle of the right thigh are pictured. Note that while not pictured, testing also required an ACE bandage be wrapped around the NIRS device to assist in securing it during testing, and a cloth be placed on top of both to obscure any additional ambient light interference.

Serial arterial occlusion technique

Initially, a 30-second cuff occlusion was performed without any NMES to assess resting muscle oxygen consumption (mVO₂). This was followed by an ischemic calibration to provide a reference point for minimum and maximum levels of oxygen saturation. This was accomplished by applying 10-seconds of NMES during a 3–5-minute arterial occlusion. Following a 3–5-minute refractory period, contractions of the target muscle were facilitated via NMES simultaneously with another cuff occlusion for 15-seconds. This aimed to deplete free energy stores within the target muscle tissue and initiate the processes necessary to replenish these substrates through mitochondrial activity. Immediately following, a series of arterial occlusions were administered to control the gradual return of oxygen. A total of 20 occlusions were applied as follows: 5-seconds on / 5-seconds off for occlusions 1–5; 5 s on/10-seconds off for occlusions 6–10; and 10-seconds on/20-seconds off for occlusions 11–20. Following another 3–5-minute refractory period, this serial arterial occlusion technique was repeated beginning with another 15-seconds of NMES.^21,22 Participant’s safety was also ensured by frequently monitoring heart rate and blood pressure throughout testing. Testing was immediately discontinued if the participant reported discomfort or any safety concerns were noted (eg.an unexpected rise in blood pressure).

Data acquisition and signal processing

The optical density (OD) of the NIRS signal was sampled at 10 Hz and transferred wirelessly through a Bluetooth module to the manufacturer’s software (Ox soft, 3.0.103.3, Artinis Medical Systems, Elst, Netherlands). O2Hb and HHb signals were then transferred to Visual 3D (6.01.22, C-motion, Germantown, MD, USA) for initial processing. Figure 2a provides representative raw NIRS signals beginning with the ischemic calibration and followed by serial arterial occlusions as previously discussed. To account for blood volume changes (it is assumed that during arterial occlusions the changes in O2Hb and HHb signals occur with a 1:1 ratio), a correction factor (β) was applied to both O2Hb and HHb signals. using the following equations:^21,22,25

$$\mathrm{\text{β}}=\left|\mathrm{O}2\mathrm{H}\mathrm{b}\right|/\left(\right|\mathrm{O}2\mathrm{H}\mathrm{b}|+|\mathrm{H}\mathrm{H}\mathrm{b}\left|\right);$$

$$\mathrm{O}2\mathrm{H}{\mathrm{b}}_{\mathrm{c}\mathrm{o}\mathrm{r}\mathrm{r}\mathrm{e}\mathrm{c}\mathrm{t}\mathrm{e}\mathrm{d}}=\mathrm{O}2\mathrm{H}\mathrm{b}-\left[\mathrm{T}\mathrm{H}\mathrm{b}{}^{\ast }\left(1-\mathrm{\text{β}}\right)\right];$$

$$\mathrm{H}\mathrm{H}{\mathrm{b}}_{\mathrm{c}\mathrm{o}\mathrm{r}\mathrm{r}\mathrm{e}\mathrm{c}\mathrm{t}\mathrm{e}\mathrm{d}}=\mathrm{H}\mathrm{H}\mathrm{b}-\left[\mathrm{T}\mathrm{H}\mathrm{b}{}^{\ast }\mathrm{\text{β}}\right].$$

Figure 2.

(a) A representative sample of NIRS testing protocol completed on an SCI participant with both the O2Hb and HHb signals displayed. Started by ischemic calibration, followed by NMES and the serial arterial occlusions. (b) Corrected O2Hb and HHb signals after applying the volume correction factor (β). (c) A representative O2Hb_corrected signal from an SCI participant’s 2nd test. Slopes of identified linear portions of the signal during occlusion are used to assess mitochondrial capacity.

Where β represents the correction factor, and both O2Hb_corrected and HHb_corrected represent corrected O2Hb and HHb signals respectively (Fig. 2b). For each arterial occlusion, mVO2 was determined by visually identifying linear portions of the slopes observed in the O2Hb_corrected signal, and then calculating the slopes of these linear components (Fig. 2c). Individual slope values for each arterial occlusion were then plotted and fit to a mono-exponential curve in JMP Pro (14.0.0; SAS Institute Inc., Cary, NC, USA). The subsequent rate constant for each curve indirectly represents mitochondrial capacity. Figure 3a shows a relatively strong curve fitting of the linear slope values for an individual with SCI.

Figure 3.

Multiple representative examples of monoexponential curves fit for SCI participants. (a) strong curve fitting (r²= 0.86) with outliers identified during a single timeframe of the test. (b) moderately strong curve fitting (r²= 0.72) with outliers randomly spread through the entire test. (c) temporal shift in the rate of change of O2Hb_corrected signal from decay (almost linear) to rise, no exponential fit was found for data points. (d) strong exponential fit (r²= 0.95) with few outliers, yet the curve continues to rise and does not reach saturation or plateau.

Results

A total of sixteen participants were recruited; however, four withdrew prior to data collection. Of the four, two participants did not show up for testing, one participant reported pain upon cuff inflation and was withdrawn by the research staff, and similarly another participant was withdrawn by the staff due to their elevated resting blood pressure. Table 1 provides demographics for the remaining twelve participants as well as calculated rate constants and r² values from the mono-exponential curve fittings. The skinfold thickness of the 12 participants was 13.8 ± 8.6 mm with a level of neurological injury that ranged from C5-T11 (Table 1).

Table 1. Physical characteristics and mitochondrial rates at baseline.

ID	Age (years)	BMI (Kg/m2)	SAT (mm)	LOI	TSI (years)	AIS	Classification	Original methods		Adjusted methods
								Rate (sec−1)	r2	Rate (sec−1)	r2
1	27	17.7	10.15	T6	4	A	Paraplegia	−0.009	0.14	0.011**	0.99**
2	41	35.3	38.25	T11	3	A	Paraplegia	–	–	–	–
3	21	16.5	12.75	T4	7	A	Paraplegia	−0.0003	0.42	–	–
4	36	20.7	10.25	T5	7	A	Paraplegia	0.013	0.31	0.010**	0.92**
5	25	15.4	5.95	C6	1	C	Tetraplegia	0.006	0.37	0.017**	0.86**
6	23	18.1	7.4	T6	1.6	A	Paraplegia	−0.002	0.22	0.018**	0.99**
8	33	30.5	13.9	T8	11	C	Paraplegia	0.002*	0.82*	0.010**	0.96**
10	51	25.3	20.35	T5	1.75	A	Paraplegia	–	–	–	–
11	44	16	15.75	C7	13	A	Tetraplegia	0.012*	0.84*	0.014**	0.90**
13	57	21.6	8.45	C5	35	A	Tetraplegia	0.002*	0.71*	0.007**	0.99**
14	20	14.2	12.05	C6	1.8	B	Tetraplegia	0.013*	0.87*	0.015**	0.93**
16	25	16.9	10.25	C7	2	B	Tetraplegia	0.053	0.34	–	–

BMI, body mass index; SAT, subcutaneous adipose tissue thickness; LOI, level of injury; TSI, time since injury; AIS, American spinal injury association impairment scale. *Originally calculated data considered likely representative of mitochondrial rates. **Data calculated using adjusted methods considered likely representative of mitochondrial rates. Rate data in the “Original Methods” column was calculated utilizing the originally described methods. Rate data in the “Adjusted Methods” section was calculated following additional methodological steps described in the text.

Data inspection during the processing and analysis phases revealed multiple irregularities and poor NIRS signal quality associated with noise and artifacts (see Fig. 4). Based on the r² values (0.71–0.87) only four out of the twelve participants (∼33%) produced data that could be considered acceptable, with an average rate constant of 0.007 s⁻¹ (ranging from 0.002–0.013 s⁻¹) (Table 1; “Original Methods” marked with “*”). Additionally, no usable data was collected from participants 2 and 10 as their subcutaneous adipose tissue (SAT) thickness was > 20 mm (the PortaMon has a thickness limit of 20 mm).

Figure 4.

A representative NIRS signal during 2 serial occlusion tests for an SCI participant; test shows multiple movement artifacts, likely due to muscle spasms, spatial displacement of the device or target tissues following cuff inflation, or some other form of signal interference. Test was interrupted and repeated due to those motion artifacts.

Given the difficulties associated with calculating the rate constants, additional methodological steps were employed to render more representative data (further explanation is described in the “Discussion”). While all the steps described in the “Methods” section were carried over into the adjusted methods, HHb_corrected signals were utilized as opposed to O2Hb_corrected signals. Additionally, clearly de-trended slope values were visually identified and removed prior to mono-exponential curve fitting in JMP Pro. Once fit, outliers were identified and removed using studentized residuals ≥2.5; and if necessary, an additional curve fitting provided final mitochondrial capacity rates.

These adjusted methods produced 50% more viable data points while also improving the curve fit for each of the rate values originally identified as usable (Table 1; “Adjusted Methods” marked with “**”). The average of these final rates was 0.013 s⁻¹ (ranging from 0.010 to 0.018 s⁻¹) with r² values ranging from 0.86 to 0.99. These methods however resulted in a loss of data points >50% for participants 3 and 16; thus, no reliable curve fitting was possible for their data.

Finally, the data from four participants were not correctly fitted even after applying the studentized residuals. The four participants had a 32% greater skin fold thickness of 20.4 ± 12.6 mm compared to the group average skinfold thickness (13.8 ± 8.6 mm). Unfortunately, data from those participants were not considered for interpretation. However, subject 3 (12.75 mm) and 16 (10.25) had SAT thickness that were less than 20 mm; suggesting that other factors may have impeded capturing or fitting NIRS recovery curve.

Discussion

The findings of the current study suggest that it is difficult to specifically identify causality for each limitation or testing error which may have led to our originally unusable and questionably reliable results. Every attempt was made to discuss possible factors that may have led to these limitations. Moreover, we have highlighted and expanded on technical and methodological considerations that may assist future researchers planning to use similar devices and protocols.

Subcutaneous adipose tissue thickness, muscle atrophy, and intramuscular fat

The maximum penetration depth for near-infrared light is known to be limited to half of the distance between the emitter and receiver optodes.²⁸ The PortaMon has three fixed emitter-receiver distances with a maximum tissue penetration depth of ∼ 20 mm. SATT above this limit prevents a substantial amount of light from reaching the target muscle tissue, confounding the assessment of skeletal muscle mitochondrial capacity. Therefore, it is highly recommended to assess SAT thickness at the target site using skinfold measurements or ultrasound imaging. It is also suggested that researchers set clear inclusion/exclusion criteria for SATT measures based on the maximal penetration depth of the used NIRS device. This is especially important when studying individuals with SCI who are characterized with increased SATT, especially in the lower extremity.¹⁰ Table 1 shows two participants (10 and 2) that deemed no usable data due to their SAT thickness exceeding the 20 mm limit. Unfortunately, such limitation cannot be corrected.

Muscle atrophy and IMF infiltration could also obstruct signal propagation through the target tissue,¹³ further limiting the reliability of NIRS signals. As light waves propagate through heterogeneous tissues with different medium properties and optical characteristics, the path of light will be obscured. As recommended, a differential pathlength factor (DPF) is applied to minimize this effect;^21,22,25 however, these values may not accurately reflect the tissue properties for those with SCI. Additionally, under-estimation of mVO₂ may occur as fat tissue has a lower metabolic rate than muscle tissue.^12,13 The use of a diagnostic ultrasound may not only provide researchers with an accurate assessment of SAT thickness, but it may also permit the assessment of muscle size/depth and IMF infiltration of the target muscle.²⁹

Muscle spasms and movement artifacts

Individuals with SCI often experience involuntary muscle movements and spasms.³⁰ This problem was often observed following cuff occlusions and/or electrical stimulation. Light wave propagation is very sensitive to spatial changes,²⁸ thus NIRS signals can be dramatically affected by involuntary movements, leading to noisy or lost data, and possibly misleading results (Fig. 4). Some studies have utilized support systems to protect the target limb against movement artifacts.^21,22,25 Variable manual support was thus provided, to safely and dynamically control involuntary limb movements. In addition, it is recommended that participants with a known history of spasticity may be encouraged to have their prescribed anti-spasmodic medications prior to testing.

Inadequate muscle stimulation

In SCI populations, consistent and controlled muscle activation is accomplished via NMES of the target muscle;^26,31 requiring correct electrode placement (as shown in Fig. 1) and accurate stimulation parameters. Previous studies suggest that a current intensity of 100–200 mA is necessary to illicit reasonable activation of the thigh musculature in persons with SCI.^26,32 The serial occlusion technique requires adequate muscle activation with repeated twitches over 10–15 s to deplete free energy stores and to stimulate their resynthesis; failure to fatigue the target muscle may likely confound the results of the test. A noticeable level of fatigue is observable when the O2Hb signal drops to ∼40–70% of the earlier ischemic occlusion test (as shown in Fig. 2a). On the contrary, over stimulation of muscle tissue may likely induce tissue injury, especially in persons with SCI who may easily experience an exercise induced muscle injury following an acute bout of exercise.^33,34 These factors underline the importance of carefully adjusting NMES parameters to accurately measure mitochondrial capacity without causing muscle injury after SCI.³²

External light interference

NIRS devices are equipped with both emitter and receiver optodes that need to be carefully applied to the target area to minimize any interaction with ambient light sources and potentially leading to measurement errors.^27,34 To achieve a high signal-to-noise ratio, it is recommended to firmly apply the optodes to the skin while also covering the device with light blocking materials (such as a black cloth), and if necessary/possible adjusting the emitter power and/or gain settings.^28,35

Ischemia induced discomfort

Few participants reported varying degrees of discomfort during arterial occlusions with high cuff pressures of 250 mmHg. While such discomfort is not entirely unexpected for even an able-bodied population, the possibility of inducing autonomic dysreflexia for SCI participants is a concern that can potentially lead to an individual’s withdrawal from participation in the study. Therefore, it is necessary to regularly monitor blood pressure and heart rate during testing to minimize the risk of encountering autonomic dysreflexia.

Sampling frequency

Sampling frequency must also be carefully chosen, as not to impact signal quality, and negatively influence results. Utilizing low signal sampling frequencies (<10 Hz) with continuous wave NIRS may lead to poor data quality and/or data loss. The PortaMon has a pre-set maximum 10 Hz sampling frequency. Sampling at higher frequencies has its own drawbacks, as the signals are often accompanied by unwanted noise; however, advanced filtering and smoothing techniques can be employed to isolate a high-fidelity signal.²⁸

Signal selection (O2Hb–HHb)

The adopted protocol for serial arterial occlusions is designed such that the researcher must select only one NIRS signal for analysis (O2Hb or HHb). Ideally, after applying the volume correction factor (β), both O2Hb and HHb signals should reflect each other.^21,22,25 Unfortunately, in a longitudinal design clinical trials, discrepancies and inconsistencies between O₂Hb and HHb signal qualities were observed; potentially caused by changes in testing conditions or data acquisition methods, or even changes in physiological status of the participant. Such signal discrepancies can impact the reliability and interpretability of the study results.

The default emitter wavelength settings for the PortaMon device were employed based on previously published studies;^21,22,25 however, at 760 nm the HHb signal has a higher discriminatory affinity when compared to 850 nm for the O2Hb signal.²⁸ This discrepancy may likely explain a tendency in the literature for researchers to select HHb signals for the assessment of mitochondrial capacity due to its relatively higher signal-to-noise ratio when compared to O2Hb.²⁸ However, other researchers have selected O2Hb signals to assess mitochondrial capacity in persons with SCI.²⁶

Automated signal processing and artifact correction

Adopting fully automated processing techniques can be challenging as artifacts due to muscle spasms and/or light interference may randomly occur. Limited published literature attempts to resolve the issue of the NIRS signal artifacts using automated methods. Sophisticated computer algorithms have been proposed that rely on the application of wavelet transformation followed by the isolation of artifacts using statistical tools.³⁶ However, these algorithms have not been validated for persons with SCI who likely experience more random muscle spasms and greater spasticity compared to able bodied individuals. Other attempts involved different hardware employing small optical fiber prisms and collodion bandages has also been observed to reduce such artifacts, but such approaches are reportedly more expensive and have only been applied in monitoring brain tissues.³⁷

Detecting the linear portions of the chosen signal during each arterial occlusion is also not easily accomplished in a fully automated fashion. Conversely, completely manual techniques can be time consuming and are subject to greater interpretation variability. We found that semi-automated data processing methods may provide the most reliable results within a reasonable time frame. This was accomplished through an algorithm that identified linear portions of the NIRS signals during arterial occlusions, followed by a visual inspection of these flagged events. Once confirmed, slope calculations and mono-exponential curve fitting were also completed by custom written code.

Mono-exponential curve fitting

Mitochondrial capacity is measured by calculating the linear slope values for corrected NIRS signal during arterial occlusions. These slope values are then plotted and fit to a mono-exponential curve where its time rate is an indication of mitochondrial capacity. Figure 3a and 3b show strong to moderate curve fitting. Outliers (likely due to limitations discussed above), were identified and removed based on studentized residual analysis with a threshold value of 2.5. Figure 3c and 3d show data with a temporal shift in the rate of change for the O2Hb_corrected signal and an unsaturated curve respectively. This may likely be due to multiple factors, such as mitochondrial dysfunction following SCI,³⁸ insufficient muscle fatigue during testing, or due to a lack of rate saturation suggesting longer occlusion protocols may be necessary for persons with SCI.^26,27 Unfortunately, such curves cannot be considered reliable leading to their exclusion and possibly requiring repetition of the test.

Conclusion

NIRS is a non-invasive, relatively inexpensive tool which can provide in vivo measurements of oxygen kinetics, and when paired with a serial arterial occlusion technique can be used to assess skeletal muscle mitochondrial capacity. Several methodological and physiological limitations may challenge the use of NIRS after SCI. Many potential considerations were provided to enhance future testing conditions, data acquisition, signal processing and analysis. Upon removal of outlying data points, we were successful in sparing data sets from being lost and improve results reliability. Current work may also provide researchers with a trouble shooting reference and guide allowing the expansion of this technique to measure mitochondrial capacity after SCI.

Abbreviations

β

beta correction factor

ATP

adenosine triphosphate

DPF

differential pathlength factor

HHb

deoxygenated hemoglobin

HHb_corrected

corrected deoxygenated hemoglobin

IMF

intramuscular fat

MRS

magnetic resonance spectroscopy

mVO2

muscle oxygen consumption

NIRS

near-infrared spectroscopy

NMES

surface neuromuscular electrical stimulation

O2Hb

oxygenated hemoglobin

O2Hb_corrected

corrected oxygenated hemoglobin

PCR

phosphocreatine

ROS

reactive oxygen species

SCI

spinal cord injury

THb

total hemoglobin

Disclaimer statements

Contributors None.

Funding This work was supported by DoD-CDRMP: [grant number W81XWH-15-1-0671]; VA RRD-CDA-2: [grant number 5IK2RX000732-05].

Ethics statement We confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.

Conflicts of interest Authors have no conflicts of interest to declare.