YMVRE-03497; No. of pages: 6; 4C: Microvascular Research xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Microvascular Research journal homepage: www.elsevier.com/locate/ymvre
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Diego Orbegozo Cortés a,1, Florin Puflea a,b,1, Katia Donadello a, Fabio Silvio Taccone a, Leonardo Gottin b, Jacques Creteur a, Jean Louis Vincent a, Daniel De Backer a,⁎
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Article history: Accepted 18 November 2014 Available online xxxx
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Keywords: Hyperoxia Microcirculation Oxygen consumption Metabolism Reactive hyperemia
Department of Intensive Care, Erasme University Hospital, Université Libre de Bruxelles, Brussels, Belgium Intensive Care Department, Azienda Ospedaliera Universitaria Integrata (AOUI), Università degli Studi di Verona, Verona, Italy
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The use of high concentrations of inhaled oxygen has been associated with adverse effects but recent data suggest a potential therapeutic role of normobaric hyperoxia (NH) in sepsis and cerebral ischemia. Hyperoxia may induce vasoconstriction and alter endothelial function, so we evaluated its effects on the microcirculation in 40 healthy adult volunteers using side-stream dark field (SDF) video-microscopy on the sublingual area and near-infrared spectroscopy (NIRS) on the thenar eminence. In a first group of volunteers (n = 18), measurements were taken every 30 min: at baseline in air, during NH (close to 100% oxygen via a non-rebreathing mask) and during recovery in air. In a second group (n = 22), NIRS measurements were taken in NH or ambient air on two separate days to prevent any potential influence of repeated NIRS measurements. NH significantly decreased the proportion of perfused vessels (PPV) from 92% to 66%, perfused vessel density (PVD) from 11.0 to 7.3 vessels/mm, small perfused vessel density (SPVD) from 9.0 to 5.8 vessels/mm and microvascular flow index (MFI) from 2.8 to 2.0, and increased PPV heterogeneity from 7.5% to 30.4%. Thirty minutes after return to air, PPV, PVD, SPVD and MFI remained partially altered. During NH, NIRS descending slope and NIRS muscle oxygen consumption (VO2) decreased from 8.5 to 7.9%/s and 127 to 103 units, respectively, in the first group and from 10.7 to 9.4%/s and 150 to 115 units in the second group. NH, therefore, alters the microcirculation in healthy subjects, decreasing capillary perfusion and VO2 and increasing the heterogeneity of the perfusion. © 2014 Published by Elsevier Inc.
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Normobaric hyperoxia alters the microcirculation in healthy volunteers☆,☆☆,★
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Introduction
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Inhalation of high concentrations of oxygen has been associated with local and systemic adverse events, including pulmonary toxicity (Crapo et al., 1983; Davis et al., 1983), reabsorption atelectasis (Santos et al., 2000), increased production of reactive oxygen species (Brueckl et al., 2006), vasoconstriction (Reinhart et al., 1991) and altered cellular metabolism (Lodato, 1989; Reinhart et al., 1991). This observation has led to the almost dogmatic concept that hyperoxia should be avoided in critically ill patients (Jenkinson, 1993). However, recent data have focused on a potential therapeutic role of elevated oxygen concentrations
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☆ Author contributions: DOC, FP, FST, JLV, DDB conceived and designed the study; DOC and FP performed the measurements; KD performed the blinded analysis of the microcirculation; DOC and FP wrote the first draft of the manuscript. DOC, FP, KD, FST, LG, JC, JLV and DDB revised the text for intellectual content. All the co-authors read and approved the final text. ☆☆ Funding: Institutional funds only. ★ Conflict of interest: The authors declare that they have no conflicts of interest related to this manuscript. ⁎ Corresponding author at: Department of Intensive Care, Erasme University Hospital, Route de Lennik 808, B-1070 Brussels, Belgium. Fax: +32 2 555 4698. E-mail address: [email protected] (D. De Backer). 1 Equal contributions.
in normobaric and hyperbaric conditions. Animal experiments using hyperoxia have shown improved neurological outcome after cerebral ischemia (Sun et al., 2011; Veltkamp et al., 2005) and a decreased inflammatory response in septic (Hauser et al., 2009; Waisman et al., 2012) and ischemia–reperfusion (Waisman et al., 2003) models. Moreover, the changes in oxygen consumption, blood lactate or other intermediate products of the Krebs cycle (Stellingwerff et al., 2005) seen during hyperoxic conditions may not be pathological and could be interpreted as an adaptive phenomenon of cellular metabolism. Oxygen is often administered to critically ill patients with the aim of improving tissue oxygenation. However, tissue oxygenation depends not only on capillary PO2 but also on microcirculatory perfusion. In normoxic conditions, microcirculatory alterations have been reported in various groups of critically ill patients, including those with sepsis (De Backer et al., 2002) and severe cardiac failure (De Backer et al., 2004), mainly represented by a decrease in various indexes of capillary perfusion and an increasing heterogeneity of perfusion. Moreover the severity of these alterations has been related to mortality of these patients (De Backer et al., 2002, 2004, 2013; den Uil et al., 2010; Sakr et al., 2004). Importantly, the diffusional component of the diseased microcirculation is more related to outcome than its convective one (Edul et al., 2012). Hence, hyperoxia could be considered as a way to
http://dx.doi.org/10.1016/j.mvr.2014.11.006 0026-2862/© 2014 Published by Elsevier Inc.
Please cite this article as: Cortés, D.O., et al., Normobaric hyperoxia alters the microcirculation in healthy volunteers, Microvasc. Res. (2014), http://dx.doi.org/10.1016/j.mvr.2014.11.006
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Methods
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After obtaining Institutional Ethical Committee approval (ref P2012/ 229), 40 healthy adult volunteers were enrolled. All participants provided written informed consent. The subjects were placed in a quiet and temperature controlled room within the department of intensive care. They rested comfortably seated for 15 min before each experiment. At least one vascular occlusion test (VOT) was performed in the previous 15 min before the initiation of measurements to familiarize the volunteers with the NIRS technology.
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Protocol
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In a first series of 18 volunteers, measurements were obtained 3 times at 30 min intervals: at baseline (PRE) breathing room air, under NH using a facial non-rebreathing mask delivering an inspired oxygen concentration close to 100%, and at recovery (POST) 30 min after returning to room air (Fig. 1). To eliminate the risk of a potential influence of the first NIRS measurement on the second one, we also studied a series of 22 volunteers on two consecutive days. On one of the days (DIRECT O2), they
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Considering the well-known response of the cardiovascular system to hyperoxemic conditions with vasoconstriction and decreasing heart rate (Lund et al., 1999; Meyer et al., 2010) we decide to evaluate noninvasively the heart rate (HR), respiratory rate (RR) and hemoglobin saturation by pulse oximetry (SpO2) with a Siemens SC 9000 monitor (Siemens, Erlangen, Germany). Non-invasive measurements of mean arterial pressure (MAP) were obtained in the opposite arm to that used for the NIRS measurements, at each study time point. Microcirculation video-microscopy recordings were performed using an SDF imaging device (Microvision Medical BV, Amsterdam, the Netherlands) with a probe on the sublingual area. At each time point, 5 different sites were evaluated and videos of at least 12 s were recorded. Measurements under NH were performed immediately after removing the face mask (similar to normoxic measurements) to avoid any potential bias related to the measurement technique at different time-points. During pilot experiments we tried sublingual microcirculation with the facemasks in place, through a lateral hole, but this was quite difficult and led to acquisition of limited quality videos. For the data analysis, all videos were placed in a randomization table and analyzed by an independent investigator (KD) blinded to the name and time of each video. A semi-quantitative analysis of the microcirculation was performed as previously described (De Backer et al., 2007). We classified vessels as small or large using a cut-off of 20 μm and characterize their flow as continuous, intermittent or absent. We then counted the number of vessels that cross three equidistant vertical and horizontal lines drawn on the screen, allowing computation of perfused vessel density (PVD), perfused small vessel density (PSVD) and proportion of perfused vessels (PPV). We then calculated the perfusion heterogeneity of PPV (PPV HI) as: (maximum PPV − minimum PPV) × 100/mean PPV. Finally we quantify the micro-vascular flow index (MFI) as absent (0), intermittent (1), sluggish (2), or normal (3) in each of the four quadrants of the video and calculated the mean value. Tissue oxygen saturation (StO2) was evaluated using a near-infrared spectrometer (InSpectra 850 model, Hutchinson Technology, Hutchinson, Minnesota), with a 15 mm-probe attached to the thenar eminence. The participant was asked not to move their hand during the procedure. When StO2 values remained stable for 1 min, we induced a VOT with a sphygmomanometer cuff wrapped around the ipsilateral arm, and inflated to a pressure of 50 mm Hg above the systolic arterial pressure for 3 min (time threshold), followed by a rapid deflation.
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were exposed to NH as described earlier for 30 min and NIRS measurements were obtained. On the other day (CONTROL DAY), measurements were performed after 30 min breathing room air (Fig. 1). The order of the study days was randomized by tossing a coin on the first day of the study.
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limit the impact of these alterations. However, the first step would be to better characterize the effects of hyperoxia on the normal microcircula71 tion. The impact of hyperoxia on the human microcirculation has not 72 been fully elucidated yet (Rousseau et al., 2007). Using intravital video 73 microscopy in experimental animals, several groups have shown a de74 crease in capillary perfusion (Bertuglia et al., 1991; Waisman et al., 2012). 75 Nowadays it is possible to evaluate human microcirculation non76 invasively. First, direct visualization of sublingual microcirculation is 77 possible using side-stream dark-field (SDF) video-microscopy. This mi78 croscope uses a green light at 530 nm that is reflected by deeper layers, 79 providing trans-illumination of the tissue and is absorbed by hemoglo80 bin, making visible red blood cells. It thus allows characterization of the 81 micro-vascular density and microcirculatory flow in the explored tissue 82 and it has been validated for human microcirculation evaluation 83 (Goedhart et al., 2007). Evaluation of oxygen content in the muscle 84 microcirculation is feasible with near-infrared spectroscopy (NIRS) 85 (Gomez et al., 2008). The emitted near infrared light is captured at a 86 distant point, and absorption characteristics of oxy- and deoxy87 hemoglobin allow their quantification in the microcirculation of the 88 explored area. During a dynamic test obtained by a transient ischemia 89 of the explored area, the speed of decrease of StO2 reflects local oxygen 90 consumption (Van Beekvelt et al., 2001) while StO2 reperfusion speed is 91 Q10 a measure of micro-vascular reactivity (Bopp et al., 2011). 92 We hypothesized that normobaric hyperoxia (NH) may alter the 93 human microcirculation, as assessed using sublingual SDF video94 microscopy and thenar eminence NIRS techniques. 69 70
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Fig. 1. Protocol design in the two cohorts of healthy volunteers.
Please cite this article as: Cortés, D.O., et al., Normobaric hyperoxia alters the microcirculation in healthy volunteers, Microvasc. Res. (2014), http://dx.doi.org/10.1016/j.mvr.2014.11.006
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Fig. 2. Individual responses of capillary perfusion (perfused vessel density [PVD]) and heterogeneity (heterogeneity index of proportion of perfused small vessels [PPV HI]) in the one-day experiment (n = 10).
Results
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Statistical analysis
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Statistical analysis was performed using SPSS 19.0 (IBM, New York, NY) software. If the variables had a normal distribution, the values are presented as means ± standard deviations, otherwise data are presented as median with percentiles (25–75%). For comparisons of values in the two-day experiment, a t-test or a Wilcoxon test was used as
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Discussion
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This is the first study to evaluate the effects of NH on the human microcirculation using a video-microscopy technique (sublingual SDF). Other groups have previously studied the microcirculation in animals using in vivo video-microscopy of the lung (Balasubramaniam et al., 2007; Waisman et al., 2012), mesentery (Waisman et al., 2003) and
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The main demographic characteristics of the volunteers are shown in Table 1. As expected, there was a significant increase in the SpO2 and a decrease in the heart rate during NH (Tables 2 and 3). Blood pressure and respiratory rate were unaltered. Venous PO2 (PvO2) and SpvO2 increased during NH while pH and PCO2 did not change (Table 4). The O2 AV dif decreased from 27.2% (11.9–47.0) to 15% (9.1–29.0) under hyperoxia and increased to 32.5% (27.0–46.4) (p b 0.05) thereafter. At baseline, all sublingual microcirculatory variables were similar to previous published data using the same technology in healthy volunteers (Donadello et al., 2011). NH was associated with a significant decrease in all indexes of capillary perfusion (PPV, PVD, PSVD, MFI) and an increase in the heterogeneity of perfusion (PPV HI) (Fig. 2). Thirty minutes after return to ambient air, all microvascular parameters had improved but PPV, PVD, PSVD and MFI did not return to baseline values (Table 5). A representative image of changes induced by NH in the sublingual microcirculation of one volunteer is shown in Fig. 4. There were similar changes in NIRS-derived variables in the two groups (Tables 6 and 7). StO2 base and StO2 max increased transiently under NH. THI only decreased significantly in the two-day experiment. The Desc slope and NIRS VO2 decreased during hyperoxia while the Asc slope remained unchanged. After NH, all NIRS parameters returned to baseline values in the one-day experiment.
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Table 1 Main characteristics of the study participants (n = 40).
Fig. 3. Evolution of StO2 during a VOT.
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InSpectra Software Analyzer version 3.0. (Hutchinson Technology) was used to measure the baseline, minimal and maximal StO2 (StO2 base, 161 StO2 min and StO2 max, respectively) and total hemoglobin index 162 (THI base, THI min and THI max, respectively). We also computed the 163 StO2 ascending (Asc) and descending (Desc) slopes (Fig. 3). We calcu164 lated the thenar muscle VO2 (NIRS VO2) using the following formula: 165 Q12 ((THI base + THI min) / 2) × −(Desc slope). 166 A peripheral venous catheter was inserted in a forearm vein, ipsilat167 eral to the NIRS probe. Before, during and after the NH test, we collected 168 2 mL of blood for blood gas analysis and co-oximetry (GEM premier 169 4000, Instrumentation Laboratory, Bedford, Massachusetts). We calcu170 lated the forearm regional arterio-venous O2 difference (AV dif) as the 171 difference between the arterial oxygen saturation (SpO2) by pulse 172 oximetry and the peripheral forearm venous oxygen saturation 173 (SpvO2) in the blood gas analysis. 174 NIRS measurements were obtained at each study time point, but 175 SDF measurements and venous blood samplings were obtained only 176 in the first 10 volunteers, because the results were already significantly 177 different in these participants.
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appropriate. For comparison at different times in the first group, a one-way analysis of variance (ANOVA) or Friedman's test was used as appropriate with corrections for repeated measurements. A 2-sided p value less than 0.05 was considered as significant for all analyses.
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Variable
Result
t1:3
Male [n (%)] Age [years ± SD] Height [cm ± SD] Weight [kg ± SD] Body mass index [kg/m2 ± SD] Hemoglobin [g/dL ± SD]
21 (52) 30 ± 6 169 ± 9 67 ± 13 23.3 ± 2.7 13.8 ± 1.8
t1:4 t1:5 t1:6 t1:7 t1:8 t1:9
Please cite this article as: Cortés, D.O., et al., Normobaric hyperoxia alters the microcirculation in healthy volunteers, Microvasc. Res. (2014), http://dx.doi.org/10.1016/j.mvr.2014.11.006
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D.O. Cortés et al. / Microvascular Research xxx (2014) xxx–xxx
Table 2 Hemodynamic changes during the one-day experiment (n = 18).
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Table 4 Forearm venous blood gas analysis from 10 volunteers.
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Variable
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HR (beats per min) MAP (mm Hg) RR (breaths per min) SpO2 (%)
77 ± 12 86 ± 10 12 ± 1 98 ± 1
72 ± 12⁎,§ 86 ± 10 12 ± 1 100 ± 0⁎,§
75 ± 12 86 ± 10 12 ± 1 98 ± 1
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⁎ p b 0.05 versus PRE. § p b 0.05 versus POST.
pH PCO2 (mm Hg) PvO2 (mm Hg) Lactate (mmol/L) HCO3 (mmol/L) SpvO2 (%)
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7.39 (7.37–7.41) 47 (40–49) 46⁎,§ (41–61) 1.0⁎ (0.8–1.1) 25.7 (25–27.3) 85.2⁎,§ (71–90.9)
7.37 (7.35–7.39) 50 (48–50) 35 (31–46) 0.8⁎ (0.6–0.8) 25.3 (24.8–27) 65.8 (52.6–72)
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Table 5 Microcirculatory variables at different time points from 10 volunteers.
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micro-vascular blood flow (MFI and capillary perfusion) and in O2 AV dif, so that VO2 must have decreased (VO2 = Flow × O2 AV dif). Moreover, these findings are in accordance with previous reports of a decrease in the global VO2 during hyperoxia, measured directly using Fick's principle in animals (Lodato, 1989) and in critically ill patients (Reinhart et al., 1991) or by indirect calorimetry in healthy volunteers (Lauscher et al., 2012). One important methodological issue is that the high concentration of dissolved oxygen in the capillaries under NH cannot be detected by NIRS. The supplemental dissolved oxygen must be consumed before the StO2 starts to decrease (Desc slope). This phenomenon should be associated with a prolonged time between initiation of the vascular occlusion and the start of the Desc slope, a period sometimes called the latency time (Gomez et al., 2008). However, identification of the inflection point of the StO2 has high inter- and intra-observer variability. Moreover, after the latency phase, the partial pressure of oxygen returns to low values again, the hemoglobin starts to become desaturated and the NIRS Desc slope (flat line) appears. At this time point, the speed of desaturation (slope) again reflects local tissue VO2 (Fig. 3). Whether reduced capillary perfusion was the consequence or the cause of the decreased VO2 we observed is a difficult question. NH may directly diminish cellular metabolism and VO2, so that the microcirculatory changes could represent just an adaptive phenomenon; alternatively, oxygen could impair the microcirculation and endothelial functioning, with subsequent metabolic alterations. Considering the first option, Stellingwerff et al. (2005, 2006) showed that exercise under hyperoxic conditions in healthy volunteers decreased muscle glycogenolysis with lower pyruvate and lactate production compared to normoxia. When hypoperfusion is present, an increase in blood lactate levels would be expected but this was not found in their experiments or ours. Another interesting fact observed during hyperoxia was that the ascending slope (a measure of microvascular reactivity) was not different from that recorded during normoxia. However, we must remember that an unchanged Asc slope just reflects the maximal response after the challenge of a VOT and does not mean that the basal flow was not altered. In reactive hyperemia conducted in rabbits, peak flow velocities were similar at different oxygen concentrations, whereas mean flow velocities or the duration of the hyperemia were different (Tuma et al., 1977). Considering the second hypothesis, the magnitude of the decrease in microvascular perfusion (decrease in PVD or PPV) was much greater than the decrease in VO2. In addition, alterations in microvascular perfusion persisted after the return to normoxia while descending slope
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skeletal muscle (Bertuglia et al., 1991). Several studies using NIRS technology showed that static values of StO2 increased under normobaric (Keramidas et al., 2012) and hyperbaric (Larsson et al., 2010) hyperoxia 220 in humans, but no studies with VOTs have been reported. Static StO2 221 values are difficult to interpret but a dynamic approach (VOTs) allows 222 non-invasive evaluation of the local muscle VO2 (Desc slope) (Van 223 Beekvelt et al., 2001) and the vascular reactivity of the microcirculation 224 Q13 (Asc slope) (Bopp et al., 2011) (Fig. 3). 225 The main finding of our investigation is that NH induces a decrease 226 in capillary perfusion, as reflected by a decrease in the PPV, perfused 227 vessel density of large and small vessels and MFI obtained by sublingual 228 video microscopy. These findings in human volunteers are concordant 229 with previous animal data that specifically reported data on the density 230 of microcirculatory perfusion (Kamler et al., 2004; Lindbom and Arfors, 231 1985; Tsai et al., 2003). Using in vivo video microscopy, the density of 232 perfused capillaries was shown to decrease under NH to around 70% 233 of baseline value in the muscle of rabbits (Lindbom and Arfors, 1985) 234 or in the dorsal microcirculation of hamsters (Kamler et al., 2004; Tsai 235 et al., 2003). 236 Control of microcirculation density is a very complex phenomenon 237 (Fry et al., 2013), and we found an approximate 30% reduction in the 238 capillary perfusion by SDF that could not be explained by a change in 239 the driving pressure into the tissues because mean arterial pressure 240 remained constant. Whether this change was accompanied by an 241 increase in vascular resistance cannot be elucidated from our data, 242 because the semi-quantitative analysis of SDF images that we 243 performed does not measure the vessel diameters. However, several 244 experimental studies using in vivo video-microscopy have addressed 245 this: Riemann et al. (2011) found a decrease in cremaster arteriolar 246 diameter in mice after exposure to hyperoxic conditions created by 247 perfusing the muscle with oxygenated red blood cells and Bertuglia 248 et al. (1991) showed that hyperoxia-induced vasoconstriction in the 249 muscle of healthy hamsters preferentially occurred in the smallest 250 vessels of the microcirculation. The decrease in MFI, which includes a 251 flow component, is in agreement with these data as micro-vascular 252 blood flow depends mainly on the diameter of the vessels (Murrant 253 et al., 2000; Fry et al., 2013). 254 Another interesting finding was the decrease in the estimated 255 muscle VO2 (Desc slope and NIRS VO2) under NH in the two groups of 256 volunteers. Several factors suggest that this was not a spurious phenom257 enon. First, NIRS VOTs have been shown to track muscle metabolic rate 258 Q14 in healthy volunteers during exercise (Boushel et al., 1998; De Blasi 259 et al., 1993; Van Beekvelt et al., 2001; Gomez et al., 2008). Of note 260 NIRS represents the measurement in a specific area of the muscle, 261 while most of the direct techniques evaluate the whole consumption 262 of one extremity, hence both values may differ but trends correlate 263 Q15 ( Boushel et al., 1998). Second, we observed a combined decrease in
NH
7.38 (7.33–7.39) 50 (45–52) 43 (31–56) 1.1 (0.9–1.5) 25.8 (23.6–27.1) 70.8 (51–87.1)
⁎ p b 0.05 versus PRE. § p b 0.05 versus POST.
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Table 3 Hemodynamic changes during the two-day experiment (n = 22).
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HR (beats per min) MAP (mm Hg) RR (breaths per min) SpO2 (%)
70 ± 6 82 ± 6 13 ± 1 99 ± 1
67 ± 7 82 ± 5 13 ± 1 100 ± 0
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PPV (%) PVD (vessel/mm) PSVD (vessel/mm) MFI (0–3) PPV HI (%)
265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306
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92.2 (91–92.5) 11.0 (10.2–12.1) 9.0 (8.4–10.6) 2.8 (2.8–2.9) 7.5 (6.1–10.5)
66.3⁎,§ (62.6–72.3) 7.3⁎,§ (5.5–8.3) 5.8⁎,§ (4.3–7.1) 2.0⁎,§ (1.8–2.3) 30.4⁎,§ (22.3–42.9)
88.4⁎ (83.5–90) 9.9⁎ (9.4–10.9) 8.1⁎ (7.2–9.3) 2.7⁎ (2.6–2.8) 10.1 (8.2–12.7)
t5:4 t5:5 t5:6 t5:7 t5:8
⁎ p b 0.05 versus PRE. § p b 0.05 versus POST.
Please cite this article as: Cortés, D.O., et al., Normobaric hyperoxia alters the microcirculation in healthy volunteers, Microvasc. Res. (2014), http://dx.doi.org/10.1016/j.mvr.2014.11.006
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D.O. Cortés et al. / Microvascular Research xxx (2014) xxx–xxx
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Table 6 NIRS variables during the one-day experiment in 18 volunteers.
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StO2 base (%) THI base (au) StO2 max (%) Desc slope (%/min) Asc slope (%/s) NIRS VO2 (au)
t6:10 t6:11
⁎ p b 0.05 versus PRE. § p b 0.05 versus POST.
t7:1 t7:2 Q5
NH
POST
79 (77–82) 14.5 (13.1–15.9) 94 (93–95) 8.5 (7.5–11.3) 4.4 (3.5–4.9) 127 (103–144)
82§ (78–82) 14 (12.6–15.4) 97⁎,§ (94–98) 7.9⁎ (6.7–9.5) 4.2 (3.5–5) 103⁎,§ (96–125)
80 (74–83) 14.5 (12.5–15.9) 95 (92–96) 8.6 (7.9–11.1) 4.1 (3.4–5.3) 119 (102–142)
Table 7 NIRS variables during the two-day experiment in 22 volunteers.
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StO2 base (%) THI base (au) StO2 max (%) Desc slope (%/min) Asc slope (%/s) NIRS VO2 (au)
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DIRECT O2
p
80 (78–83) 15 (13.9–15.7) 94 (93–95) 10.7 (9.1–12.1) 4.5 (4.1–5.2) 150 (133–174)
83 (81–86) 14 (12.6–15.4) 97 (95–98) 9.4 (7.5–11.1) 4.7 (3.9–5.3) 115 (102–139)
0.02 0.01 b0.01 0.02 0.84 b0.01
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(Suzuki et al., 1997) with an increased neutrophil adherence (Bowman et al., 1983) and macrophage activation (Horinouchi et al., 1996). Once these processes are started, they do not disappear immediately after removal of the stimulus. Several studies have indicated that the damage is increased with longer exposures to NH (Crapo et al., 1983; Waisman et al., 2012), but short periods may not be completely without harm. The potential therapeutic use of NH in specific conditions, such as brain injury, depends on the balance between benefit and harm and the actual data on the potential harm of NH on the microcirculation is limited. Finally, we must remark that our study was designed to evaluate the effects of NH on the microcirculation, but it is very limited to detect the physio-pathological pathways involved in the appearance of these changes. It would be needed to obtain additional invasive and direct measurements of blood flow and VO2, as measurement of different inflammatory pathways and probably invasively control blood flow as in the study of Bredle et al. (1988). However, we decided to be as less invasive as possible to make easier the recruitment of volunteers. To definitively determine if these changes are adaptive or not, additional research in the field is necessary. In conclusion, NH did induce a decrease in capillary perfusion and in muscle VO2, and increased microcirculatory perfusion heterogeneity. NH did not alter the reactive hyperemia after an ischemic challenge. These microcirculatory variables have been independently associated with patient outcomes, including mortality (Sakr et al., 2004), so they should be further evaluated during the use of hyperoxia as a therapeutic strategy.
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normalized at that stage. These two findings suggest that changes in microvascular perfusion were not just following VO2 changes. We also observed a marked increase in the heterogeneity of capillary perfusion a condition that has been typically associated with the distributive alterations observed in sepsis and associated with alterations in tissue perfusion and markers of severity in critically ill patients (Edul et al., 2012; Trzeciak et al., 2007). Under normal conditions, microcirculatory adaptations that couple a decrease in metabolic demand would not increase heterogeneity (Humer et al., 1996; Walley, 1996). More importantly, Bredle et al. (1988) perfused isolated hind-limb muscles in a dog model and demonstrated that the decrease in muscle VO2 while perfusing hyperoxic autologous blood was explained by the maldistribution of blood flow. When these investigators kept the driving pressure constant (flow depends on vascular resistance), muscle VO2 decreased during hyperoxia, but remained normal or elevated when the hind limb flow was kept constant. Taken together, these data suggest that the alterations in microvascular perfusion were not adaptive to a decrease in VO2 but rather induced it. It was unexpected that micro-vascular perfusion failed to return to baseline values 30 min after the return to normoxia, even though muscle VO2 had returned to baseline. We could not find any data regarding the time needed to resolve microcirculatory alterations after short and acute exposure to NH. Several studies have shown that under hyperoxic conditions there is an increase in capillary production of reactive oxygen species (Brueckl et al., 2006), a modification in nitric oxide pathways (Xu et al., 2002) and an increase in endothelial adherence factors
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Fig. 4. Representative sublingual SDF video-microscopy images in one volunteer at baseline (A) and under NH (B). There was a decrease in quantity of vessels that cross the grid (white lines) and the appearance of an altered flow in some vessels (arrow).
Please cite this article as: Cortés, D.O., et al., Normobaric hyperoxia alters the microcirculation in healthy volunteers, Microvasc. Res. (2014), http://dx.doi.org/10.1016/j.mvr.2014.11.006
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Meyer, C., Rana, O.R., Saygili, E., Ozüyaman, B., Latz, K., Rassaf, T., Kelm, M., Schauerte, P., 2010. Hyperoxic chemoreflex sensitivity is impaired in patients with neurocardiogenic syncope. Int. J. Cardiol. 142 (1), 38–43. Murrant, C.L., Sarelius, I.H., 2000. Coupling of muscle metabolism and muscle blood flow in capillary units during contraction. Acta Physiol. Scand. 168 (4), 531–541. Reinhart, K., Bloos, F., Konig, F., Bredle, D., Hannemann, L., 1991. Reversible decrease of oxygen consumption by hyperoxia. Chest 99, 690–694. Riemann, M., Rai, A., Ngo, A.T., Dziegiel, M.H., Holstein-Rathlou, N.H., Torp-Pedersen, C., 2011. Oxygen-dependent vasomotor responses are conducted upstream in the mouse cremaster microcirculation. J. Vasc. Res. 48, 79–89. Rousseau, A., Steinwall, I., Woodson, R.D., Sjoberg, F., 2007. Hyperoxia decreases cutaneous blood flow in high-perfusion areas. Microvasc. Res. 74, 15–22. Sakr, Y., Dubois, M.J., De Backer, D., Creteur, J., Vincent, J.L., 2004. Persistent microcirculatory alterations are associated with organ failure and death in patients with septic shock. Crit. Care Med. 32, 1825–1831. Santos, C., Ferrer, M., Roca, J., Torres, A., Hernandez, C., Rodriguez-Roisin, R., 2000. Pulmonary gas exchange response to oxygen breathing in acute lung injury. Am. J Respir. Crit. Care Med. 161, 26–31. Stellingwerff, T., Glazier, L., Watt, M.J., LeBlanc, P.J., Heigenhauser, G.J., Spriet, L.L., 2005. Effects of hyperoxia on skeletal muscle carbohydrate metabolism during transient and steady-state exercise. J. Appl. Physiol. 98, 250–256. Stellingwerff, T., LeBlanc, P.J., Hollidge, M.G., Heigenhauser, G.J., Spriet, L.L., 2006. Hyperoxia decreases muscle glycogenolysis, lactate production, and lactate efflux during steady-state exercise. Am. J. Physiol. Endocrinol. Metab. 290, E1180–E1190. Sun, L., Strelow, H., Mies, G., Veltkamp, R., 2011. Oxygen therapy improves energy metabolism in focal cerebral ischemia. Brain Res. 1415, 103–108. Suzuki, Y., Aoki, T., Takeuchi, O., Nishio, K., Suzuki, K., Miyata, A., Oyamada, Y., Takasugi, T., Mori, M., Fujita, H., Yamaguchi, K., 1997. Effect of hyperoxia on adhesion molecule expression in human endothelial cells and neutrophils. Am. J. Physiol. 272, L418–L425. Trzeciak, S., Dellinger, R.P., Parrillo, J.E., Guglielmi, M., Bajaj, J., Abate, N.L., Arnold, R.C., Colilla, S., Zanotti, S., Hollenberg, S.M., 2007. Early microcirculatory perfusion derangements in patients with severe sepsis and septic shock: relationship to hemodynamics, oxygen transport, and survival. Ann. Emerg. Med. 49, 88–98 (98). Tsai, A.G., Cabrales, P., Winslow, R.M., Intaglietta, M., 2003. Microvascular oxygen distribution in awake hamster window chamber model during hyperoxia. Am. J. Physiol. Heart Circ. Physiol. 285, H1537–H1545. Tuma, R.F., Lindbom, L., Arfors, K.E., 1977. Dependence of reactive hyperemia in skeletal muscle on oxygen tension. Am. J. Physiol. 233, H289–H294. Van Beekvelt, M.C., Colier, W.N., Wevers, R.A., Van Engelen, B.G., 2001. Performance of near-infrared spectroscopy in measuring local O(2) consumption and blood flow in skeletal muscle. J. Appl. Physiol. (1985) 90, 511–519. Veltkamp, R., Siebing, D.A., Sun, L., Heiland, S., Bieber, K., Marti, H.H., Nagel, S., Schwab, S., Schwaninger, M., 2005. Hyperbaric oxygen reduces blood–brain barrier damage and edema after transient focal cerebral ischemia. Stroke 36, 1679–1683. Waisman, D., Brod, V., Wolff, R., Sabo, E., Chernin, M., Weintraub, Z., Rotschild, A., Bitterman, H., 2003. Effects of hyperoxia on local and remote microcirculatory inflammatory response after splanchnic ischemia and reperfusion. Am. J. Physiol. Heart Circ. Physiol. 285, H643–H652. Waisman, D., Brod, V., Rahat, M.A., mit-Cohen, B.C., Lahat, N., Rimar, D., Menn-Josephy, H., David, M., Lavon, O., Cavari, Y., Bitterman, H., 2012. Dose-related effects of hyperoxia on the lung inflammatory response in septic rats. Shock 37, 95–102. Walley, K.R., 1996. Heterogeneity of oxygen delivery impairs oxygen extraction by peripheral tissues: theory. J. Appl. Physiol. 81 (2), 885–894. Xu, F., Fok, T.F., Yung, E., Yang, M., Yin, J.A., 2002. Endothelial and inducible nitric oxide synthase gene and protein expression in hyperoxia-induced lung injury in premature rat. Acta Pharmacol. Sin. 23, 52–58 (Suppl.).
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Contents lists available at ScienceDirect
Microvascular Research journal homepage: www.elsevier.com/locate/ymvre
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Diego Orbegozo Cortés a,1, Florin Puflea a,b,1, Katia Donadello a, Fabio Silvio Taccone a, Leonardo Gottin b, Jacques Creteur a, Jean Louis Vincent a, Daniel De Backer a,⁎
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Article history: Accepted 18 November 2014 Available online xxxx
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Keywords: Hyperoxia Microcirculation Oxygen consumption Metabolism Reactive hyperemia
Department of Intensive Care, Erasme University Hospital, Université Libre de Bruxelles, Brussels, Belgium Intensive Care Department, Azienda Ospedaliera Universitaria Integrata (AOUI), Università degli Studi di Verona, Verona, Italy
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The use of high concentrations of inhaled oxygen has been associated with adverse effects but recent data suggest a potential therapeutic role of normobaric hyperoxia (NH) in sepsis and cerebral ischemia. Hyperoxia may induce vasoconstriction and alter endothelial function, so we evaluated its effects on the microcirculation in 40 healthy adult volunteers using side-stream dark field (SDF) video-microscopy on the sublingual area and near-infrared spectroscopy (NIRS) on the thenar eminence. In a first group of volunteers (n = 18), measurements were taken every 30 min: at baseline in air, during NH (close to 100% oxygen via a non-rebreathing mask) and during recovery in air. In a second group (n = 22), NIRS measurements were taken in NH or ambient air on two separate days to prevent any potential influence of repeated NIRS measurements. NH significantly decreased the proportion of perfused vessels (PPV) from 92% to 66%, perfused vessel density (PVD) from 11.0 to 7.3 vessels/mm, small perfused vessel density (SPVD) from 9.0 to 5.8 vessels/mm and microvascular flow index (MFI) from 2.8 to 2.0, and increased PPV heterogeneity from 7.5% to 30.4%. Thirty minutes after return to air, PPV, PVD, SPVD and MFI remained partially altered. During NH, NIRS descending slope and NIRS muscle oxygen consumption (VO2) decreased from 8.5 to 7.9%/s and 127 to 103 units, respectively, in the first group and from 10.7 to 9.4%/s and 150 to 115 units in the second group. NH, therefore, alters the microcirculation in healthy subjects, decreasing capillary perfusion and VO2 and increasing the heterogeneity of the perfusion. © 2014 Published by Elsevier Inc.
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Introduction
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Inhalation of high concentrations of oxygen has been associated with local and systemic adverse events, including pulmonary toxicity (Crapo et al., 1983; Davis et al., 1983), reabsorption atelectasis (Santos et al., 2000), increased production of reactive oxygen species (Brueckl et al., 2006), vasoconstriction (Reinhart et al., 1991) and altered cellular metabolism (Lodato, 1989; Reinhart et al., 1991). This observation has led to the almost dogmatic concept that hyperoxia should be avoided in critically ill patients (Jenkinson, 1993). However, recent data have focused on a potential therapeutic role of elevated oxygen concentrations
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☆ Author contributions: DOC, FP, FST, JLV, DDB conceived and designed the study; DOC and FP performed the measurements; KD performed the blinded analysis of the microcirculation; DOC and FP wrote the first draft of the manuscript. DOC, FP, KD, FST, LG, JC, JLV and DDB revised the text for intellectual content. All the co-authors read and approved the final text. ☆☆ Funding: Institutional funds only. ★ Conflict of interest: The authors declare that they have no conflicts of interest related to this manuscript. ⁎ Corresponding author at: Department of Intensive Care, Erasme University Hospital, Route de Lennik 808, B-1070 Brussels, Belgium. Fax: +32 2 555 4698. E-mail address: [email protected] (D. De Backer). 1 Equal contributions.
in normobaric and hyperbaric conditions. Animal experiments using hyperoxia have shown improved neurological outcome after cerebral ischemia (Sun et al., 2011; Veltkamp et al., 2005) and a decreased inflammatory response in septic (Hauser et al., 2009; Waisman et al., 2012) and ischemia–reperfusion (Waisman et al., 2003) models. Moreover, the changes in oxygen consumption, blood lactate or other intermediate products of the Krebs cycle (Stellingwerff et al., 2005) seen during hyperoxic conditions may not be pathological and could be interpreted as an adaptive phenomenon of cellular metabolism. Oxygen is often administered to critically ill patients with the aim of improving tissue oxygenation. However, tissue oxygenation depends not only on capillary PO2 but also on microcirculatory perfusion. In normoxic conditions, microcirculatory alterations have been reported in various groups of critically ill patients, including those with sepsis (De Backer et al., 2002) and severe cardiac failure (De Backer et al., 2004), mainly represented by a decrease in various indexes of capillary perfusion and an increasing heterogeneity of perfusion. Moreover the severity of these alterations has been related to mortality of these patients (De Backer et al., 2002, 2004, 2013; den Uil et al., 2010; Sakr et al., 2004). Importantly, the diffusional component of the diseased microcirculation is more related to outcome than its convective one (Edul et al., 2012). Hence, hyperoxia could be considered as a way to
http://dx.doi.org/10.1016/j.mvr.2014.11.006 0026-2862/© 2014 Published by Elsevier Inc.
Please cite this article as: Cortés, D.O., et al., Normobaric hyperoxia alters the microcirculation in healthy volunteers, Microvasc. Res. (2014), http://dx.doi.org/10.1016/j.mvr.2014.11.006
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Methods
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After obtaining Institutional Ethical Committee approval (ref P2012/ 229), 40 healthy adult volunteers were enrolled. All participants provided written informed consent. The subjects were placed in a quiet and temperature controlled room within the department of intensive care. They rested comfortably seated for 15 min before each experiment. At least one vascular occlusion test (VOT) was performed in the previous 15 min before the initiation of measurements to familiarize the volunteers with the NIRS technology.
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Protocol
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In a first series of 18 volunteers, measurements were obtained 3 times at 30 min intervals: at baseline (PRE) breathing room air, under NH using a facial non-rebreathing mask delivering an inspired oxygen concentration close to 100%, and at recovery (POST) 30 min after returning to room air (Fig. 1). To eliminate the risk of a potential influence of the first NIRS measurement on the second one, we also studied a series of 22 volunteers on two consecutive days. On one of the days (DIRECT O2), they
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Considering the well-known response of the cardiovascular system to hyperoxemic conditions with vasoconstriction and decreasing heart rate (Lund et al., 1999; Meyer et al., 2010) we decide to evaluate noninvasively the heart rate (HR), respiratory rate (RR) and hemoglobin saturation by pulse oximetry (SpO2) with a Siemens SC 9000 monitor (Siemens, Erlangen, Germany). Non-invasive measurements of mean arterial pressure (MAP) were obtained in the opposite arm to that used for the NIRS measurements, at each study time point. Microcirculation video-microscopy recordings were performed using an SDF imaging device (Microvision Medical BV, Amsterdam, the Netherlands) with a probe on the sublingual area. At each time point, 5 different sites were evaluated and videos of at least 12 s were recorded. Measurements under NH were performed immediately after removing the face mask (similar to normoxic measurements) to avoid any potential bias related to the measurement technique at different time-points. During pilot experiments we tried sublingual microcirculation with the facemasks in place, through a lateral hole, but this was quite difficult and led to acquisition of limited quality videos. For the data analysis, all videos were placed in a randomization table and analyzed by an independent investigator (KD) blinded to the name and time of each video. A semi-quantitative analysis of the microcirculation was performed as previously described (De Backer et al., 2007). We classified vessels as small or large using a cut-off of 20 μm and characterize their flow as continuous, intermittent or absent. We then counted the number of vessels that cross three equidistant vertical and horizontal lines drawn on the screen, allowing computation of perfused vessel density (PVD), perfused small vessel density (PSVD) and proportion of perfused vessels (PPV). We then calculated the perfusion heterogeneity of PPV (PPV HI) as: (maximum PPV − minimum PPV) × 100/mean PPV. Finally we quantify the micro-vascular flow index (MFI) as absent (0), intermittent (1), sluggish (2), or normal (3) in each of the four quadrants of the video and calculated the mean value. Tissue oxygen saturation (StO2) was evaluated using a near-infrared spectrometer (InSpectra 850 model, Hutchinson Technology, Hutchinson, Minnesota), with a 15 mm-probe attached to the thenar eminence. The participant was asked not to move their hand during the procedure. When StO2 values remained stable for 1 min, we induced a VOT with a sphygmomanometer cuff wrapped around the ipsilateral arm, and inflated to a pressure of 50 mm Hg above the systolic arterial pressure for 3 min (time threshold), followed by a rapid deflation.
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were exposed to NH as described earlier for 30 min and NIRS measurements were obtained. On the other day (CONTROL DAY), measurements were performed after 30 min breathing room air (Fig. 1). The order of the study days was randomized by tossing a coin on the first day of the study.
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limit the impact of these alterations. However, the first step would be to better characterize the effects of hyperoxia on the normal microcircula71 tion. The impact of hyperoxia on the human microcirculation has not 72 been fully elucidated yet (Rousseau et al., 2007). Using intravital video 73 microscopy in experimental animals, several groups have shown a de74 crease in capillary perfusion (Bertuglia et al., 1991; Waisman et al., 2012). 75 Nowadays it is possible to evaluate human microcirculation non76 invasively. First, direct visualization of sublingual microcirculation is 77 possible using side-stream dark-field (SDF) video-microscopy. This mi78 croscope uses a green light at 530 nm that is reflected by deeper layers, 79 providing trans-illumination of the tissue and is absorbed by hemoglo80 bin, making visible red blood cells. It thus allows characterization of the 81 micro-vascular density and microcirculatory flow in the explored tissue 82 and it has been validated for human microcirculation evaluation 83 (Goedhart et al., 2007). Evaluation of oxygen content in the muscle 84 microcirculation is feasible with near-infrared spectroscopy (NIRS) 85 (Gomez et al., 2008). The emitted near infrared light is captured at a 86 distant point, and absorption characteristics of oxy- and deoxy87 hemoglobin allow their quantification in the microcirculation of the 88 explored area. During a dynamic test obtained by a transient ischemia 89 of the explored area, the speed of decrease of StO2 reflects local oxygen 90 consumption (Van Beekvelt et al., 2001) while StO2 reperfusion speed is 91 Q10 a measure of micro-vascular reactivity (Bopp et al., 2011). 92 We hypothesized that normobaric hyperoxia (NH) may alter the 93 human microcirculation, as assessed using sublingual SDF video94 microscopy and thenar eminence NIRS techniques. 69 70
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Fig. 1. Protocol design in the two cohorts of healthy volunteers.
Please cite this article as: Cortés, D.O., et al., Normobaric hyperoxia alters the microcirculation in healthy volunteers, Microvasc. Res. (2014), http://dx.doi.org/10.1016/j.mvr.2014.11.006
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Fig. 2. Individual responses of capillary perfusion (perfused vessel density [PVD]) and heterogeneity (heterogeneity index of proportion of perfused small vessels [PPV HI]) in the one-day experiment (n = 10).
Results
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Statistical analysis was performed using SPSS 19.0 (IBM, New York, NY) software. If the variables had a normal distribution, the values are presented as means ± standard deviations, otherwise data are presented as median with percentiles (25–75%). For comparisons of values in the two-day experiment, a t-test or a Wilcoxon test was used as
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Discussion
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This is the first study to evaluate the effects of NH on the human microcirculation using a video-microscopy technique (sublingual SDF). Other groups have previously studied the microcirculation in animals using in vivo video-microscopy of the lung (Balasubramaniam et al., 2007; Waisman et al., 2012), mesentery (Waisman et al., 2003) and
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The main demographic characteristics of the volunteers are shown in Table 1. As expected, there was a significant increase in the SpO2 and a decrease in the heart rate during NH (Tables 2 and 3). Blood pressure and respiratory rate were unaltered. Venous PO2 (PvO2) and SpvO2 increased during NH while pH and PCO2 did not change (Table 4). The O2 AV dif decreased from 27.2% (11.9–47.0) to 15% (9.1–29.0) under hyperoxia and increased to 32.5% (27.0–46.4) (p b 0.05) thereafter. At baseline, all sublingual microcirculatory variables were similar to previous published data using the same technology in healthy volunteers (Donadello et al., 2011). NH was associated with a significant decrease in all indexes of capillary perfusion (PPV, PVD, PSVD, MFI) and an increase in the heterogeneity of perfusion (PPV HI) (Fig. 2). Thirty minutes after return to ambient air, all microvascular parameters had improved but PPV, PVD, PSVD and MFI did not return to baseline values (Table 5). A representative image of changes induced by NH in the sublingual microcirculation of one volunteer is shown in Fig. 4. There were similar changes in NIRS-derived variables in the two groups (Tables 6 and 7). StO2 base and StO2 max increased transiently under NH. THI only decreased significantly in the two-day experiment. The Desc slope and NIRS VO2 decreased during hyperoxia while the Asc slope remained unchanged. After NH, all NIRS parameters returned to baseline values in the one-day experiment.
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Table 1 Main characteristics of the study participants (n = 40).
Fig. 3. Evolution of StO2 during a VOT.
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InSpectra Software Analyzer version 3.0. (Hutchinson Technology) was used to measure the baseline, minimal and maximal StO2 (StO2 base, 161 StO2 min and StO2 max, respectively) and total hemoglobin index 162 (THI base, THI min and THI max, respectively). We also computed the 163 StO2 ascending (Asc) and descending (Desc) slopes (Fig. 3). We calcu164 lated the thenar muscle VO2 (NIRS VO2) using the following formula: 165 Q12 ((THI base + THI min) / 2) × −(Desc slope). 166 A peripheral venous catheter was inserted in a forearm vein, ipsilat167 eral to the NIRS probe. Before, during and after the NH test, we collected 168 2 mL of blood for blood gas analysis and co-oximetry (GEM premier 169 4000, Instrumentation Laboratory, Bedford, Massachusetts). We calcu170 lated the forearm regional arterio-venous O2 difference (AV dif) as the 171 difference between the arterial oxygen saturation (SpO2) by pulse 172 oximetry and the peripheral forearm venous oxygen saturation 173 (SpvO2) in the blood gas analysis. 174 NIRS measurements were obtained at each study time point, but 175 SDF measurements and venous blood samplings were obtained only 176 in the first 10 volunteers, because the results were already significantly 177 different in these participants.
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appropriate. For comparison at different times in the first group, a one-way analysis of variance (ANOVA) or Friedman's test was used as appropriate with corrections for repeated measurements. A 2-sided p value less than 0.05 was considered as significant for all analyses.
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Male [n (%)] Age [years ± SD] Height [cm ± SD] Weight [kg ± SD] Body mass index [kg/m2 ± SD] Hemoglobin [g/dL ± SD]
21 (52) 30 ± 6 169 ± 9 67 ± 13 23.3 ± 2.7 13.8 ± 1.8
t1:4 t1:5 t1:6 t1:7 t1:8 t1:9
Please cite this article as: Cortés, D.O., et al., Normobaric hyperoxia alters the microcirculation in healthy volunteers, Microvasc. Res. (2014), http://dx.doi.org/10.1016/j.mvr.2014.11.006
191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210
214 215 216
4 t2:1 t2:2
D.O. Cortés et al. / Microvascular Research xxx (2014) xxx–xxx
Table 2 Hemodynamic changes during the one-day experiment (n = 18).
t4:1 Q1 t4:2
Table 4 Forearm venous blood gas analysis from 10 volunteers.
t2:3
Variable
PRE
NH
POST
t2:4 t2:5 t2:6 t2:7
HR (beats per min) MAP (mm Hg) RR (breaths per min) SpO2 (%)
77 ± 12 86 ± 10 12 ± 1 98 ± 1
72 ± 12⁎,§ 86 ± 10 12 ± 1 100 ± 0⁎,§
75 ± 12 86 ± 10 12 ± 1 98 ± 1
t2:8 t2:9
⁎ p b 0.05 versus PRE. § p b 0.05 versus POST.
pH PCO2 (mm Hg) PvO2 (mm Hg) Lactate (mmol/L) HCO3 (mmol/L) SpvO2 (%)
POST
t4:3
7.39 (7.37–7.41) 47 (40–49) 46⁎,§ (41–61) 1.0⁎ (0.8–1.1) 25.7 (25–27.3) 85.2⁎,§ (71–90.9)
7.37 (7.35–7.39) 50 (48–50) 35 (31–46) 0.8⁎ (0.6–0.8) 25.3 (24.8–27) 65.8 (52.6–72)
t4:4 t4:5 t4:6 t4:7 t4:8 t4:9 t4:10 t4:11
264
Table 5 Microcirculatory variables at different time points from 10 volunteers.
t5:1 Q2 t5:2
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micro-vascular blood flow (MFI and capillary perfusion) and in O2 AV dif, so that VO2 must have decreased (VO2 = Flow × O2 AV dif). Moreover, these findings are in accordance with previous reports of a decrease in the global VO2 during hyperoxia, measured directly using Fick's principle in animals (Lodato, 1989) and in critically ill patients (Reinhart et al., 1991) or by indirect calorimetry in healthy volunteers (Lauscher et al., 2012). One important methodological issue is that the high concentration of dissolved oxygen in the capillaries under NH cannot be detected by NIRS. The supplemental dissolved oxygen must be consumed before the StO2 starts to decrease (Desc slope). This phenomenon should be associated with a prolonged time between initiation of the vascular occlusion and the start of the Desc slope, a period sometimes called the latency time (Gomez et al., 2008). However, identification of the inflection point of the StO2 has high inter- and intra-observer variability. Moreover, after the latency phase, the partial pressure of oxygen returns to low values again, the hemoglobin starts to become desaturated and the NIRS Desc slope (flat line) appears. At this time point, the speed of desaturation (slope) again reflects local tissue VO2 (Fig. 3). Whether reduced capillary perfusion was the consequence or the cause of the decreased VO2 we observed is a difficult question. NH may directly diminish cellular metabolism and VO2, so that the microcirculatory changes could represent just an adaptive phenomenon; alternatively, oxygen could impair the microcirculation and endothelial functioning, with subsequent metabolic alterations. Considering the first option, Stellingwerff et al. (2005, 2006) showed that exercise under hyperoxic conditions in healthy volunteers decreased muscle glycogenolysis with lower pyruvate and lactate production compared to normoxia. When hypoperfusion is present, an increase in blood lactate levels would be expected but this was not found in their experiments or ours. Another interesting fact observed during hyperoxia was that the ascending slope (a measure of microvascular reactivity) was not different from that recorded during normoxia. However, we must remember that an unchanged Asc slope just reflects the maximal response after the challenge of a VOT and does not mean that the basal flow was not altered. In reactive hyperemia conducted in rabbits, peak flow velocities were similar at different oxygen concentrations, whereas mean flow velocities or the duration of the hyperemia were different (Tuma et al., 1977). Considering the second hypothesis, the magnitude of the decrease in microvascular perfusion (decrease in PVD or PPV) was much greater than the decrease in VO2. In addition, alterations in microvascular perfusion persisted after the return to normoxia while descending slope
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skeletal muscle (Bertuglia et al., 1991). Several studies using NIRS technology showed that static values of StO2 increased under normobaric (Keramidas et al., 2012) and hyperbaric (Larsson et al., 2010) hyperoxia 220 in humans, but no studies with VOTs have been reported. Static StO2 221 values are difficult to interpret but a dynamic approach (VOTs) allows 222 non-invasive evaluation of the local muscle VO2 (Desc slope) (Van 223 Beekvelt et al., 2001) and the vascular reactivity of the microcirculation 224 Q13 (Asc slope) (Bopp et al., 2011) (Fig. 3). 225 The main finding of our investigation is that NH induces a decrease 226 in capillary perfusion, as reflected by a decrease in the PPV, perfused 227 vessel density of large and small vessels and MFI obtained by sublingual 228 video microscopy. These findings in human volunteers are concordant 229 with previous animal data that specifically reported data on the density 230 of microcirculatory perfusion (Kamler et al., 2004; Lindbom and Arfors, 231 1985; Tsai et al., 2003). Using in vivo video microscopy, the density of 232 perfused capillaries was shown to decrease under NH to around 70% 233 of baseline value in the muscle of rabbits (Lindbom and Arfors, 1985) 234 or in the dorsal microcirculation of hamsters (Kamler et al., 2004; Tsai 235 et al., 2003). 236 Control of microcirculation density is a very complex phenomenon 237 (Fry et al., 2013), and we found an approximate 30% reduction in the 238 capillary perfusion by SDF that could not be explained by a change in 239 the driving pressure into the tissues because mean arterial pressure 240 remained constant. Whether this change was accompanied by an 241 increase in vascular resistance cannot be elucidated from our data, 242 because the semi-quantitative analysis of SDF images that we 243 performed does not measure the vessel diameters. However, several 244 experimental studies using in vivo video-microscopy have addressed 245 this: Riemann et al. (2011) found a decrease in cremaster arteriolar 246 diameter in mice after exposure to hyperoxic conditions created by 247 perfusing the muscle with oxygenated red blood cells and Bertuglia 248 et al. (1991) showed that hyperoxia-induced vasoconstriction in the 249 muscle of healthy hamsters preferentially occurred in the smallest 250 vessels of the microcirculation. The decrease in MFI, which includes a 251 flow component, is in agreement with these data as micro-vascular 252 blood flow depends mainly on the diameter of the vessels (Murrant 253 et al., 2000; Fry et al., 2013). 254 Another interesting finding was the decrease in the estimated 255 muscle VO2 (Desc slope and NIRS VO2) under NH in the two groups of 256 volunteers. Several factors suggest that this was not a spurious phenom257 enon. First, NIRS VOTs have been shown to track muscle metabolic rate 258 Q14 in healthy volunteers during exercise (Boushel et al., 1998; De Blasi 259 et al., 1993; Van Beekvelt et al., 2001; Gomez et al., 2008). Of note 260 NIRS represents the measurement in a specific area of the muscle, 261 while most of the direct techniques evaluate the whole consumption 262 of one extremity, hence both values may differ but trends correlate 263 Q15 ( Boushel et al., 1998). Second, we observed a combined decrease in
NH
7.38 (7.33–7.39) 50 (45–52) 43 (31–56) 1.1 (0.9–1.5) 25.8 (23.6–27.1) 70.8 (51–87.1)
⁎ p b 0.05 versus PRE. § p b 0.05 versus POST.
217 218 219
PRE
t3:1 t3:2
Table 3 Hemodynamic changes during the two-day experiment (n = 22).
t3:3
Variable
CONTROL DAY
DIRECT O2
p
t3:4 t3:5 t3:6 t3:7
HR (beats per min) MAP (mm Hg) RR (breaths per min) SpO2 (%)
70 ± 6 82 ± 6 13 ± 1 99 ± 1
67 ± 7 82 ± 5 13 ± 1 100 ± 0
b0.01 0.19 0.38 b0.01
PPV (%) PVD (vessel/mm) PSVD (vessel/mm) MFI (0–3) PPV HI (%)
265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306
PRE
NH
POST
t5:3
92.2 (91–92.5) 11.0 (10.2–12.1) 9.0 (8.4–10.6) 2.8 (2.8–2.9) 7.5 (6.1–10.5)
66.3⁎,§ (62.6–72.3) 7.3⁎,§ (5.5–8.3) 5.8⁎,§ (4.3–7.1) 2.0⁎,§ (1.8–2.3) 30.4⁎,§ (22.3–42.9)
88.4⁎ (83.5–90) 9.9⁎ (9.4–10.9) 8.1⁎ (7.2–9.3) 2.7⁎ (2.6–2.8) 10.1 (8.2–12.7)
t5:4 t5:5 t5:6 t5:7 t5:8
⁎ p b 0.05 versus PRE. § p b 0.05 versus POST.
Please cite this article as: Cortés, D.O., et al., Normobaric hyperoxia alters the microcirculation in healthy volunteers, Microvasc. Res. (2014), http://dx.doi.org/10.1016/j.mvr.2014.11.006
t5:9 t5:10
D.O. Cortés et al. / Microvascular Research xxx (2014) xxx–xxx
5
322 323 324 325 326 327 328 329 330 331 332
t6:1 t6:2 Q3
Table 6 NIRS variables during the one-day experiment in 18 volunteers.
t6:3
StO2 base (%) THI base (au) StO2 max (%) Desc slope (%/min) Asc slope (%/s) NIRS VO2 (au)
t6:10 t6:11
⁎ p b 0.05 versus PRE. § p b 0.05 versus POST.
t7:1 t7:2 Q5
NH
POST
79 (77–82) 14.5 (13.1–15.9) 94 (93–95) 8.5 (7.5–11.3) 4.4 (3.5–4.9) 127 (103–144)
82§ (78–82) 14 (12.6–15.4) 97⁎,§ (94–98) 7.9⁎ (6.7–9.5) 4.2 (3.5–5) 103⁎,§ (96–125)
80 (74–83) 14.5 (12.5–15.9) 95 (92–96) 8.6 (7.9–11.1) 4.1 (3.4–5.3) 119 (102–142)
Table 7 NIRS variables during the two-day experiment in 22 volunteers.
t7:3 t7:4 t7:5 t7:6 t7:7 t7:8 t7:9
PRE
U
t6:4 t6:5 t6:6 t6:7 t6:8 t6:9 Q4
StO2 base (%) THI base (au) StO2 max (%) Desc slope (%/min) Asc slope (%/s) NIRS VO2 (au)
CONTROL DAY
DIRECT O2
p
80 (78–83) 15 (13.9–15.7) 94 (93–95) 10.7 (9.1–12.1) 4.5 (4.1–5.2) 150 (133–174)
83 (81–86) 14 (12.6–15.4) 97 (95–98) 9.4 (7.5–11.1) 4.7 (3.9–5.3) 115 (102–139)
0.02 0.01 b0.01 0.02 0.84 b0.01
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(Suzuki et al., 1997) with an increased neutrophil adherence (Bowman et al., 1983) and macrophage activation (Horinouchi et al., 1996). Once these processes are started, they do not disappear immediately after removal of the stimulus. Several studies have indicated that the damage is increased with longer exposures to NH (Crapo et al., 1983; Waisman et al., 2012), but short periods may not be completely without harm. The potential therapeutic use of NH in specific conditions, such as brain injury, depends on the balance between benefit and harm and the actual data on the potential harm of NH on the microcirculation is limited. Finally, we must remark that our study was designed to evaluate the effects of NH on the microcirculation, but it is very limited to detect the physio-pathological pathways involved in the appearance of these changes. It would be needed to obtain additional invasive and direct measurements of blood flow and VO2, as measurement of different inflammatory pathways and probably invasively control blood flow as in the study of Bredle et al. (1988). However, we decided to be as less invasive as possible to make easier the recruitment of volunteers. To definitively determine if these changes are adaptive or not, additional research in the field is necessary. In conclusion, NH did induce a decrease in capillary perfusion and in muscle VO2, and increased microcirculatory perfusion heterogeneity. NH did not alter the reactive hyperemia after an ischemic challenge. These microcirculatory variables have been independently associated with patient outcomes, including mortality (Sakr et al., 2004), so they should be further evaluated during the use of hyperoxia as a therapeutic strategy.
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normalized at that stage. These two findings suggest that changes in microvascular perfusion were not just following VO2 changes. We also observed a marked increase in the heterogeneity of capillary perfusion a condition that has been typically associated with the distributive alterations observed in sepsis and associated with alterations in tissue perfusion and markers of severity in critically ill patients (Edul et al., 2012; Trzeciak et al., 2007). Under normal conditions, microcirculatory adaptations that couple a decrease in metabolic demand would not increase heterogeneity (Humer et al., 1996; Walley, 1996). More importantly, Bredle et al. (1988) perfused isolated hind-limb muscles in a dog model and demonstrated that the decrease in muscle VO2 while perfusing hyperoxic autologous blood was explained by the maldistribution of blood flow. When these investigators kept the driving pressure constant (flow depends on vascular resistance), muscle VO2 decreased during hyperoxia, but remained normal or elevated when the hind limb flow was kept constant. Taken together, these data suggest that the alterations in microvascular perfusion were not adaptive to a decrease in VO2 but rather induced it. It was unexpected that micro-vascular perfusion failed to return to baseline values 30 min after the return to normoxia, even though muscle VO2 had returned to baseline. We could not find any data regarding the time needed to resolve microcirculatory alterations after short and acute exposure to NH. Several studies have shown that under hyperoxic conditions there is an increase in capillary production of reactive oxygen species (Brueckl et al., 2006), a modification in nitric oxide pathways (Xu et al., 2002) and an increase in endothelial adherence factors
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Fig. 4. Representative sublingual SDF video-microscopy images in one volunteer at baseline (A) and under NH (B). There was a decrease in quantity of vessels that cross the grid (white lines) and the appearance of an altered flow in some vessels (arrow).
Please cite this article as: Cortés, D.O., et al., Normobaric hyperoxia alters the microcirculation in healthy volunteers, Microvasc. Res. (2014), http://dx.doi.org/10.1016/j.mvr.2014.11.006
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