Supplementary material

Materials and methods

LBNP protocol

The lower body of participants was comfortably positioned supine in a negative pressure chamber and an airtight seal was applied at the level of the iliac crest (Figure S1). As described in detail by Goswami and colleagues, the application site of the seal and technical considerations are of prime importance [27-28]. The LBNP protocol consisted of a baseline period of 30 min followed by stepwise manipulation of negative pressure from 0 to -20, -40, -60, -80, and -100 mmHg. The LBNP protocol could immediately be terminated by the subject or attending physician in case of impending cardiovascular collapse or per request of the subject. Impending cardiovascular collapse was defined as a drop in systolic blood pressure of >15 mmHg from baseline blood pressure, sudden bradycardia, or as subject-requested termination due to dizziness, nausea, or discomfort. Cardiovascular and NIRS parameters were recorded at the end of each LBNP step and just before the onset of cardiovascular collapse. Data obtained during cardiovascular collapse were excluded from the analysis as this study focused mainly on the early detection of hypovolemia when physiological compensatory mechanisms were still adequate.

Blood shift monitoring

To monitor the shift of blood volume away from the upper body, thoracic bioimpedance (NICOM, Cheetah Medical Inc., Wilmington, DE) was used to measure thoracic fluid content (TFC) which was expected to decrease when blood is shifted away from the upper body. The electrodes were placed on the thorax according to the manufacturer’s instructions. The TFC measurement using the NICOM device has been intensively validated by Squara et al. and Raval et al. [13-15].

Additionally, to monitor the shift of blood volume towards the lower body, a single-depth 15 mm NIRS probe (InSpectra, Hutchinson Technology, Hutchinson, MN) was placed on the medial soleus of the left calf to monitor the calf THI which was expected to increase when blood is shifted towards the lower body [9].

Hemodynamic monitoring

To monitor the physiological changes associated with LBNP, CO, SV, HR, mean arterial pressure (MAP), and SVR were continuously and non-invasively measured using volume-clamp finger plethysmography (Nexfin monitor, BMEYE, Amsterdam, the Netherlands). The Nexfin monitor finger cuff was placed on the middle phalanx of the right middle finger, which was kept at heart level. Nexfin and its predecessors (Finapres®, Finometer®, TNO-TPD Biomedical Instrumentation, Amsterdam, the Netherlands) have been extensively validated and have been shown to provide reliable measurements of cardiovascular parameters [16-18].

Multi-site and multi-depth NIRS

Multi-depth NIRS was employed to measure StO2 and THI in the thenar and forearm during LBNP. Both the StO2 and the THI calculation from the NIRS signal have been validated by Myers et al. [19-20].

Two multi-depth NIRS devices (InSpectra, Hutchinson Technology, Hutchinson, MN) were used to measure StO2 and THI continuously and non-invasively in the left thenar eminence and in the lateral side of the anterior surface of the left forearm, as described previously [11,21]. The spectrometers use reflectance mode probes with a 1.5 mm optical fiber to illuminate tissue and three 0.4 mm optical fibers, spaced 2.5, 15, and 25 mm from the illuminating fiber, to collect backscattered light from multiple (i.e., 2.5, 15, and 25 mm) and differential (i.e., 15-2.5 and 25-2.5 mm) depths. As described by Chance et al. [22] and Cui et al. [23], the NIRS probing depth increases with increasing distance between the illumination and detection fibers. Thus, by subtracting the spectral absorbance measured by the 2.5 mm fiber from that measured by the 15 and 25 mm fibers, the spectral absorbance of the skin (as measured by the fiber at 2.5 mm) was suppressed and the spectral absorbance of underlying tissue (as measured by the fibers at 15 and 25 mm) was determined. We refer to the NIRS measurements performed on the forearm and thenar with the 15 mm and 25 mm probe spacing as F15, F25, T15, and T25, respectively.

Results

LBNP-induced blood volume shift and stroke volume reduction

All 24 subjects completed the LBNP=-20 and -40 mmHg steps, 23 subjects completed the LBNP=-60 mmHg step, 15 subjects completed the LBNP=-80 mmHg step, and 9 subjects completed the LBNP=-100 mmHg step.

In order to assess the blood volume shift during LBNP, we monitored TFC in combination with calf THI. Figure 1 shows that in all subjects, TFC decreased significantly (P<0.05) from 51±2 AU at bsln to 45±2 and 44±2 AU (87±1 and 81±1 % of bsln) at LBNP=-60 and -100 mmHg, respectively.

Calf THI increased significantly (P<0.05) from 8.5±0.5 AU at bsln to 9.3±0.5 and 10.1±1.0 AU (110±2 and 113±5 % of bsln) at LBNP=-60 and -100 mmHg, respectively. The decreased upper body blood volume was associated with a reduction in SV as shown Figure 1. Results showed a significant decrease (P<0.05) in SV from 116±3 mL at bsln to 74±2 and 60±2 mL (64±1 and 50±2 % of bsln) at LBNP=-60 and -100 mmHg, respectively. Hence, calf THI was effective in demonstrating a shift in blood volume from the thorax to the lower body.

As the decrease in SV consequent to LBNP was the primary trigger for compensatory mechanisms activation, data were categorized according to SV=100 % of bsln (n=24), SV=100-80 % of bsln (n=36), SV=80-60 % of bsln (n=34), and SV=60-40 % (n=25).

Hemodynamic response

In response to the decrease in SV, HR and SVR increased. At mild central hypovolemia induced by LBNP, HR remained unchanged (P>0.05). When hypovolemia progressed, HR increased significantly (P<0.05) from 65±2 beats/min (108±2 % of bsln) at SV=100-80 % of bsln to 80±2 beats/min (136±3 % of bsln) at SV= 80-60 % of bsln, and 108±3 beats/min (179±6 % of bsln) at SV= 60-40 % of bsln. SVR increased significantly (P<0.05) from 1063±37 AU at bsln to 1162±26 AU (110±2 % of bsln) at SV=100-80 % of bsln. SVR remained unchanged (P>0.05) during further reduction of SV. During the entire LBNP protocol, with exclusion of cardiovascular collapse, CO and MAP maintained around baseline level (P>0.05), hence reflecting adequate compensation by increased HR and SVR.

Combined, these cardiovascular results confirm that LBNP indeed resulted in a significant shift of blood volume (i.e., decreased TFC and increased calf THI) and led to a significant decrease in SV. Hence, the presented LBNP setup serves as a suitable model for studying the efficacy of NIRS for the detection of (compensated) hypovolemia.

Multi-site and multi-depth NIRS

To monitor changes in peripheral tissue oxygenation (StO2) and hemoglobin content (THI), multi-depth NIRS was applied on the forearm and thenar. All NIRS results, expressed as percentage of baseline value, are shown in Figure 2. In general, forearm NIRS measurements are more sensitive to reflect changes in SV than thenar NIRS measurements and the sensitivity of these measurements does not depend on the NIRS probing depth. Where HR did not show a significant change in the first SV category of SV=100-80 % of bsln, forearm and thenar StO2 and THI did for both probing depths. However, changes measured on the thenar were of marginal extent. In the first SV category, forearm StO2 decreased significantly (P<0.05) from 77±2 % at bsln to 73±2 % (F15, 94±1 % of bsln) and 82±1% to 79±1% (F25, 95±1 % of bsln) and forearm THI decreased significantly (P<0.05) from 6.2±0.4 to 5.8±0.3 AU (F15, 91±1 % of bsln) and from 12.6±0.7 to 12.1±0.6 AU (F25, 92±1 % of bsln). At the lowest SV category of SV=60-40 % of bsln, forearm StO2 decreased significantly (P<0.05) to 68±3 % (F15, 87±2 % of bsln) and 74±2% (F25, 89±2 % of bsln) and forearm THI decreased significantly (P<0.05) to 5.3±0.4 AU (F15, 84±2 % of bsln) and 10.7±0.7 AU (F25, 85±1 % of bsln). Correlation analysis showed significant (p<0.05) positive correlations for the forearm StO2 [% of blsn] and THI [% of blsn] versus SV [% of blsn]: Pearson’s r = 0.55, 0.47, 0.67, and 0.52 for F15 StO2, F25 StO2, F15 THI, and F25 THI, respectively.

Figure S1. The lower body negative pressure setup with the subject comfortably positioned with the lower body in the negative pressure chamber. The three near-infrared spectroscopy devices for thenar, forearm, and calf measurements are depicted on the left.