Short intermittent hypoxic exposures augment ventilation but do not alter regional cerebral and muscle oxygenation during hypoxic exercise

Respiratory Physiology & Neurobiology 181 (2012) 132–142

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Short intermittent hypoxic exposures augment ventilation but do not alter regional cerebral and muscle oxygenation during hypoxic exercise Tadej Debevec a,b,∗ , Igor B. Mekjavic a a b

Department of Automation, Biocybernetics and Robotics, “Jozef Stefan” Institute, Ljubljana, Slovenia Jozef Stefan International Postgraduate School, Ljubljana, Slovenia

a r t i c l e

i n f o

Article history: Accepted 23 February 2012 Keywords: Normobaric hypoxia Exercise ventilation Near infrared spectroscopy Oxygenation

a b s t r a c t This study investigated the effects of four exposures to normobaric hypoxia (SIH group; FIO2 = 0.120, N = 10) or placebo-control normoxia (Control group; FIO2 = 0.209, N = 9) on cardio-respiratory responses to hypoxic exercise. Before and after the exposures all subjects performed a constant power test (CP) to exhaustion in hypoxia (FIO2 = 0.120) at a work load corresponding to 75% of previously determined normoxic V˙ O2 peak . Arterial oxygen saturation (SpO2 ) and minute ventilation (V˙ E ) were measured continuously. NIRS was used to monitor regional changes in oxygenated, de-oxygenated and total hemoglobin concentrations of the frontal cortex, vastus lateralis and serratus anterior. Although neither group improved CP time, the SIH group exhibited increases in both V˙ E (+15%; P < 0.05) and SpO2 (+4%; P < 0.05) after intermittent hypoxia. No physiologically significant differences were observed during exercise in vastus lateralis, serratus anterior and cerebral oxygenation between groups and testing periods. These data suggest that normobaric SIH enhances hypoxic exercise V˙ E and SpO2 , without affecting regional oxygenation or time to exhaustion. © 2012 Elsevier B.V. All rights reserved.

1. Introduction It has been reported, that intermittent hypoxic exposures can augment normoxic and/or hypoxic endurance performance (Bonetti and Hopkins, 2009; Muza, 2007). However, the minimal hypoxic dose, needed to induce favorable adaptations, remains indefinite. While the protocols employing longer hypoxic exposures (≥12 h per day) mainly base their efficiency on hematological benefits (Levine and Stray-Gundersen, 2005), the protocols using shorter exposures (SIH) may also enhance performance primarily through ventilatory adaptations (Beidleman et al., 2008). Augmented hypoxic chemo-sensitivity (Mahamed and Duffin, 2001; Sheel and Macnutt, 2008), resulting in increased exercise ventilation (V˙ E ) and arterial oxygen saturation (SpO2 ) (Katayama et al., 2001; Levine et al., 1992; Ricart et al., 2000) seems to be one of the main underlying factors for SIH induced improvements in hypoxic performance. Resting (Foster et al., 2005; Garcia et al., 2000) and exercise (Katayama et al., 2001; Ricart et al., 2000) ventilatory responses to hypoxia have been shown to increase significantly after only a few short exposures to hypoxia with

∗ Corresponding author at: Department of Automation, Biocybernetics and Robotics, Jozef Stefan Institute, Jamova 39, SI-1000, Ljubljana, Slovenia. Tel.: +386 1 4773 777; fax: +386 1 4773 154. E-mail address: [email protected] (T. Debevec). 1569-9048/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.resp.2012.02.008

no significant effect of either the duration or activity during the hypoxic exposures (Kupper and Schoffl, 2009; Muza et al., 2010). Whereas hypobaric SIH protocols have yielded favorable results in terms of ventilatory acclimatization (Katayama et al., 2001) and hypoxic performance improvement (Beidleman et al., 2003, 2008), very few studies investigated the effects of normobaric SIH on subsequent hypoxic performance metrics (Muza, 2007). These studies have shown a significant ventilatory acclimatization as reflected in increased resting SpO2 and a hypoxic ventilatory response under both hypobaric (Beidleman et al., 2009; Fulco et al., 2011) or normobaric hypoxic condition (Garcia et al., 2000; Katayama et al., 2005, 2007). Based on the above, we hypothesized that four exposures to severe normobaric hypoxia (FIO2 = 0.120) on four subsequent days would induce ventilatory adaptations resulting in enhanced exercise ventilatory response in hypoxia. Secondly, we hypothesized that the SIH mediated increase in the work of breathing (i.e. increased ventilation) would affect oxygenation modulation and blood volume distribution between the respiratory and leg muscles during high intensity hypoxic exercise. Namely, Harms et al. (1997) suggested that a significant redistribution of oxygen flux occurs during high energy demands of the respiratory muscles, that have been shown to significantly influence the locomotor fatigue and subsequent exercise performance in acute hypoxia (Amann et al., 2007). At heavy exercise loads (≥80% V˙ O2 peak ) the increased work of breathing can, even in untrained subjects with lower absolute minute ventilation levels, affect endurance performance through the blood flow

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redistribution from the exercising limbs to the respiratory muscles (Legrand et al., 2007). Moreover, exercise hyperventilation has been shown to result in a hypocapnia mediated cerebral vasoconstriction leading to diminished cerebral blood flow (Gonzalez-Alonso et al., 2004; Imray et al., 2005). We therefore hypothesized that the SIH induced increased in V˙ E would also result in a significantly increased cerebral de-oxygenation during high intensity hypoxic exercise. The main aim of this study was to determine whether four exposures to severe normobaric hypoxia can induce beneficial ventilatory adaptations for subsequent exercise performance in normobaric hypoxia and moreover, if these adaptation result in regional oxygenation and/or blood volume changes in the cerebral tissue or leg and respiratory muscles. The tested hypotheses were therefore: (1) SIH induces ventilatory adaptations resulting in enhanced hypoxic exercise ventilatory response, (2) SIH induced ventilatory adaptations alter blood distribution and oxygenation pattern between the respiratory and working leg muscle and (3) exercise hyperventilation following SIH results in a greater cerebral de-oxygenation at high exercise intensities. 2. Materials and methods 2.1. Subjects Active, healthy and non-smoking male subjects were recruited for participation in the study. All applicants, low altitude (∼300 m) residents, were specifically screened for absence of asthma, hematological and kidney disorders and altitude residence (>2500 m) within the preceding three months. Nineteen subjects meeting the above criteria were upon selection randomly assigned to either the SIH (n = 10, age 22.9 ± 1.6 years, stature 180 ± 3 cm, body mass 74.2 ± 4.9 kg, body fat 9.5 ± 4.4%, V˙ O peak 2

48.8 ± 7.5 mL kg−1 min−1 ) or the Control (n = 9, age 22.3 ± 3.4 years, stature 179 ± 3 cm, body mass 77.4 ± 11.2 kg, body fat 11.2 ± 4.6%, V˙ O2 peak 47.3 ± 5.1 mL kg−1 min−1 ) group. No significant differences in the observed physical and cardio-respiratory variables were noted between groups. Prior to participation in the study all subjects signed their written informed consent, completed separate health and training questionnaires and were familiarized with the study protocol, as well as the risks involved. 2.2. Experimental design The study was performed as a randomized, single blind, placebo controlled design with one independent factor (SIH vs. Control) and two repeated measures; trial (PRE vs. POST) and treatment (hypoxia vs. normoxia (sham)). The study protocol consisted of two testing trials and four intermittent exposures in-between. Both groups participated in four daily exposures in a climatic chamber maintained at either normobaric hypoxic (SIH group) or normobaric normoxic (Control group) condition. Exercise tests were performed the day before (PRE) and the day after (POST) the intermittent exposures. The subjects were requested to refrain from strenuous exercise for at least 24 h before the exercise tests and during the duration of the 6-day protocol. On both testing days (PRE and POST) each subject underwent a graded exercise test (V˙ O2 peak ) under normoxic condition in the morning and a constant power test (CP) under hypoxic condition in the afternoon, with a minimal 6-h interval between the tests. All exercise tests were performed in the same laboratory (Jozef Stefan Institute, Ljubljana, Slovenia), located at 300-m altitude under constant environmental conditions (air temperature = 22 ± 1.5 ◦ C; humidity = 40 ± 7%; ambient pressure = 978.2 ± 6.4 h Pa). Subjects’ anthropometric characteristics (height, mass) were measured before the intermittent exposures

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and percentage body fat was calculated from nine skin folds measurements. POST tests were performed during the same hours of the day as for the PRE tests. The experimental protocol was approved by the National Ethics Committee at Ministry of Health and conducted according to the Helsinki declaration guidelines. The outline of the study design is presented in Fig. 1. 2.3. Experimental procedures 2.3.1. Intermittent hypoxic exposures The intermittent hypoxic protocol was performed on four subsequent days. Each day subjects resided for 4 h in an environmental chamber (IZR d.o.o., Skofja Loka, Slovenia). During the exposures, the chamber was maintained at a temperature of 24 ◦ C, and relative humidity of 50%. For the SIH group, the normobaric hypoxic environment (FIO2 = 0.120 ± 0.05; equivalent air altitude = 4200 m) within the chamber was maintained using a Vacuum Pressure Swing Adsorption system (b-Cat, Tiel, The Netherlands), that generated and delivered the oxygen-depleted air to the chamber. The chamber air was continuously sampled and analyzed for O2 and CO2 content. The mean CO2 levels in the chamber did not exceed 0.05%. For the Control group the FIO2 was kept constant at 0.209. During the exposures heart rate (HR) and SpO2 were recorded at a 2min intervals. The subjects were naïve regarding the exposure and were unable to observe any of the FIO2 , HR and SpO2 readings. Subjects completed a self assessment portion of a Lake Louise Mountain sickness questionnaire (0–15) following each exposure session, to obtain the individual Lake Louise score (LLS) (Kayser et al., 2010). 2.3.2. Graded exercise test The subjects performed a graded exercise test to voluntary exhaustion on electromagnetically controlled cycle-ergometer Ergo Bike Premium (Daum electronics, Fürth, Germany) in the morning of each testing day. Resting and exercise cardiorespiratory responses were measured using a Quark CPET metabolic cart (Cosmed, Rome, Italy). During the test the subjects breathed through an oro-nasal mask (Vmask, Hans Rudolph, Shawnee, USA), connected to a two-way valve. The turbine flowmeter and gas analyzer were calibrated before each test using a 3-L syringe and two different gas mixtures, respectively. The testing protocol commenced with a 5-min resting period, followed by a 3-min warm up at a work rate of 60 W. Thereafter the workload was increased each minute by 30 W. The subjects were required to maintain a cadence of 60 min−1 throughout the whole test and were given strong verbal encouragement. The test was terminated when the subject was unable to maintain the assigned cadence. In addition, a plateau in oxygen uptake and a respiratory exchange ratio (RER) > 1.1 were used to confirm the attainment of the peak oxygen consumption (V˙ O2 peak ). V˙ O2 peak was defined as the highest 60-s average of oxygen uptake (V˙ O ) over the course of the test. Peak 2

power output (Wmax ) was calculated using the following equation: Wmax = Wcompl + (t/60 × 30). Wcompl corresponds to the last completed workload and t corresponds to the number of seconds during final uncompleted workload. 2.3.3. Constant power test to exhaustion in hypoxia The CP test was during each testing period performed in the afternoon. Prior to the CP test, subjects performed a short 5 min warm-up at a work load ranging from 40 to 90 W, on an electromagnetically braked cycloergometer model Ergo Bike Premium (Daum electronics, Fürth, Germany). The test comprised a 3min rest period in normoxia, followed by a 3-min rest period breathing a hypoxic gas mixture. Thereafter the subjects performed a 2-min warm up followed immediately by an increase in work rate to an individually pre-determined level. The individual

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Fig. 1. Study design. Subjects in the control (CON) and short intermittent hypoxic (SIH) exposure groups participated in 4 trials (days 2–5) during which they breathed room air (CON) or a hypoxic gas mixture (SIH) for 4 h. Before (PRE) and after (POST) the 4 trials hypoxic constant power (CP) tests were conducted.

absolute work rate was identical to the work load attained at 75% of V˙ O2 peak measured during the graded exercise test. During the tests, with the exception of the initial warm-up and a 3-min rest period, subjects breathed a humidified hypoxic gas mixture (FIO2 = 0.120; FIN2 = 0.880) from a 200 L Douglas bag. The subjects breathed through a two way low resistance valve (Model 2, 700 T-Shape, Hans Rudolph, Shawnee, USA) and were instructed to maintain their cycling cadence between 60 and 70 rpm. If the cadence was increased above 70 rpm the subjects were immediately verbally cautioned to reduce their cadence. The failure to maintain a cycling cadence above 60 min−1 , following an initial

verbal warning, resulted in termination of the test. The final score of the test was determined as the number of seconds a subject was able to maintain the assigned cycling cadence. Subjects reported their ratings of perceived exertion (RPE) using an adjusted (0–10) Borg scale. They reported RPE separately for the dyspnea (RPEdys ) and leg (RPEleg ) sensation. Quantification of respiratory frequency (fR ) and tidal volume (VT ) along with the continuous breath-by-breath measurements of V˙ O2 , carbon dioxide output (V˙ CO2 ), end-tidal carbon dioxide partial pressure (PETCO2 ) and minute ventilation (V˙ E ) were performed in the same manner, using the same equipment described for the graded exercise test.

Fig. 2. Individual and group (means ± SD) relative percentage changes in minute ventilation (V˙ E ; upper panel) and arterial oxygen saturation (SpO2 , lower panel) during the POST hypoxic constant power test compared to the PRE test for both groups. Values are plotted as a function of relative exercise time. WU: warm up.

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Throughout the entire test HR and SpO2 were continuously measured using a portable heart rate monitor (Polar S810i, Kempele, Finland) and a portable finger pulse oxymetry device (Nellcor, BCI 3301, Boulder, USA) with ±2 units accuracy across the range of 70–100% (Langton and Hanning, 1990), respectively. Strong verbal encouragement from the experimental personnel was given during the whole test. 2.3.4. NIRS measurement Near infrared spectroscopy (NIRS) enables continuous real time tissue oxygenation measurement during rest and exercise (Ferrari et al., 2004). However, only few studies have to date utilized it for monitoring changes following different hypoxic training modalities (Marshall et al., 2008). Given that NIRS allows for monitoring oxygenation and blood volume during exercise, it can be used for assessing the balance of muscle oxygen demand and supply (Legrand et al., 2007). During the CP tests, subjects were instrumented with three pairs of NIRS probes to continuously monitor light absorption across cerebral and muscle tissue (Oxymon MK III, Artinis Medical systems, Zatten, Netherlands). The cerebral probes were placed over the left frontal cortex region. Leg muscle probes were positioned over the distal belly of the right vastus lateralis muscle, 15 cm above the knee and 5 cm laterally from the tight midline. The fat layer on the leg measurement location, as measured with a calibrated caliper, was 10.1 ± 3.3 mm for the SIH and 9.8 ± 3.1 mm for the Control group. The probes were also positioned on the right serratus anterior muscle, fixed in the sixth intercostal space on the anterior auxiliary line. The measurements of accessory respiratory muscles oxygenation has been shown to be useful, for assessment of respiratory work, as they play an important role in maintaining ventilation levels when the diaphragm (primary respiratory muscle) is fatigued (Johnson et al., 1993). Elastic bandages were used to preclude any external light source affecting the optodes. Probe positioning and stabilization techniques were performed according to the previously published reports using the same device (Legrand et al., 2007; Subudhi et al., 2007). The instrument recorded and calculated the micromolar changes in tissue oxy-hemoglobin ([O2 Hb]) and deoxy-hemoglobin ([HHb]) across time. The changes were calculated from two NIR light wavelengths (780 and 850 nm) according to the Beer–Lambert law and using an age dependent differential path length factors (DPF; range 4.95–6.06) adjustment (Duncan et al., 1995). The sum of [O2 Hb] and [HHb] defined the total hemoglobin ([tHb]) that can be used as an index of regional blood volume changes (Van Beekvelt et al., 2001). All measurements were normalized to reflect the changes from the resting period in normoxia prior to the initiation of exercise. The data were recorded at 125 Hz and were filtered using Moving Gaussian algorithm prior to analysis. 2.3.5. Hematological tests Blood samples were collected from the antecubital vein on the morning of both PRE and POST tests (Fig. 1). Subjects were fasted prior to the procedure. All blood samples were assayed by a clinical biochemical laboratory (AdriaLab, Sinlab Group, Ljubljana, Slovenia). The samples were analyzed for red blood cell count (RBC), hemoglobin (Hb) and hematocrit (Hct), using the cytochemical impedance method (Pentra120; Horiba ABX Diagnostics, Montpellier, France) (CV < 2%). Transferrin and ferritin were analyzed using the immunoturbidimetrical method (Cobas 6000, Roche Diagnostics, Basel, Switzerland) (CV < 2.6%). 2.4. Analyses Anthropometrical characteristics and cardio-respiratory variables (V˙ O2 peak , Wmax , and V˙ Emax ) obtained during the graded exercise test before and after the protocol were compared using

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Table 1 Average values of the measured hematological variables at PRE and POST testing of both groups. SIH group PRE −12

−1

RBC (10 L ) Hct (%) −1 Hb (g L ) Transferrin (g L−1 ) Ferritin (ng mL−1 )

4.9 0.44 150 1.9 129

CON group POST

± ± ± ± ±

0.4 0.03 9 0.3 60

4.9 0.44 151 2.1 117

PRE ± ± ± ± ±

0.3 0.02 9 0.3 53

4.9 0.44 148 2.3 126

POST ± ± ± ± ±

0.2 0.03 10 0.5 65

4.8 0.43 147 2.3 124

± ± ± ± ±

0.3 0.03 9 0.4 59

Values are means ± SD. RBC, red blood cell count; Hct, hematocrit; Hb, hemoglobin.

a Student’s two-tailed t-tests. Differences between group means in CP tests, over the experimental period were analyzed using a 2-way ANOVA [group (SIH, Control) × testing period (PRE, POST)]. Three way ANOVA was used to compare the effects of the tested protocol during the CP tests in both groups across both relative (rest, 20, 40, 60, 80, 100% CP time) and absolute (rest, 1, 2, 3, 4 min) times and across PRE and POST testing [group (SIH, Control) × work load (relative, absolute) × testing period (PRE, POST)]. Where a main effect was observed a Tukey’s post hoc test was used to compare the specific differences. Multiple regression was used to calculate correlations between selected parameters. Criterion level for significance was set a priori at P < 0.05. The data are presented as means ± SD unless otherwise indicated. All analyses were performed using Statistica 5.0 (StatSoft, Inc., Tulsa, USA). 3. Results 3.1. Intermittent exposures Significant differences were observed between groups in SpO2 during the SIH exposures (1. exp.: 82 ± 3%, 2. exp.: 83 ± 2%, 3. exp.: 83 ± 4%, 4. exp.: 84 ± 3%) and Control (1. exp.: 97 ± 2%, 2. exp.: 97 ± 3%, 3. exp.: 97 ± 2%, 4. exp.: 97 ± 2%) trials. The average HR levels during the intermittent exposures were higher, although not significantly, in the SIH group compared to the Control (SIH [1. exp.: 69 ± 10, 2. exp.: 69 ± 8, 3. exp.: 66 ± 9, 4. exp.: 68 ± 8 (beats min−1 )], Control [1. exp.: 64 ± 8, 2. exp.: 64 ± 10, 3. exp.: 65 ± 7, 4. exp.: 67 ± 10 (beats min−1 )]). The LLS values were <0.7 during all exposures in the SIH group and <0.5 in the Control group. No adverse side effects associated with the hypoxic exposure (nausea, headache, dizziness) were noted. When interviewed at the end of experiment all subjects in both groups perceived their exposure as being hypoxic and not normoxic. 3.2. Hematological parameters There were no significant differences in hematological parameters between both groups and testing trials (Table 1). 3.3. Graded exercise and CP tests No differences were noted in the cardio-respiratory variables measured during the graded exercise test in normoxia following the protocol in neither group. The average PRE–POST values were: V˙ O2 peak (SIH: 48.8 ± 7.5 to 48.9 ± 6.9 mL kg−1 min−1 ; Control: 47.3 ± 5.1 to 47.1 ± 5.9 mL kg−1 min−1 ), Wmax (SIH: 322 ± 43 to 323 ± 35 W; Control: 330 ± 40 to 326 ± 37 W) and V˙ Emax (SIH: 128 ± 16 to 132 ± 24 L min−1 ; Control: 130 ± 18 to 127 ± 19 L min−1 ). The mean pre-calculated hypoxic CP test work rate was 243 ± 26 for the SIH and 245 ± 23 for the Control group. Time to exhaustion during the hypoxic CP test was not significantly different in neither group following the protocol (SIH [PRE = 295 ± 115 s,

75 ± 7 75 ± 7 77 ± 5 2

Statistically significant changes from PRE (P < 0.05). *

78 ± 4 85 ± 3 92 ± 1 73 ± 8 74 ± 7 76 ± 6 80 ± 5 84 ± 4

2

92 ± 1

± ± ± ± 84 2.4 35.3 37.2 22 0.4 5.6 3.9 ± ± ± ± 55 2.2 23.4 38.6 12 0.5 5.8 5.5 ± ± ± ± 16 1.0 14.7 34.4 31 0.6 7.5 3.5 ± ± ± ± 129 2.5 50.9 31.8 33 0.6 6.1 3.8 ± ± ± ± 116 2.8 41.8 35.2 25 0.5 6.7 4.1 ± ± ± ± 103 2.7 37.9 37.3 15 0.5 4.4 3.6 ± ± ± ± 84 2.7 30.6 39.9 13 0.5 3.5 2.9 ± ± ± ± 48 2.2 21.7 40.9 7 0.3 3.2 1.7 ± ± ± ±

Values are means ± SD. V˙ E , expired ventilation; VT , tidal volume; fR , respiratory frequency; PETCO , end-tidal carbon dioxide partial pressure; SpO2 , arterial oxygen saturation.

± ± ± ± 113 2.5 43.8 33.8 27 0.5 7.8* 3.5

100 ± 27 2.5 ± 0.5 40.1 ± 7.3 36.1 ± 3.0

80% 60% 40% 20% REST

CON group POST

100% 80% 60% 40% 20% REST

CON group PRE

11 0.8 13.3 35.5 V˙ E (L) VT (L) fR (breaths min−1 ) PETCO (mmHg)

SpO2 (%)

27 0.4 6.7 2.6 125 2.4 50.8 31.2 28 0.5 7.4 2.8

100%

± ± ± ±

79 ± 8* 78 ± 8 80 ± 7* 83 ± 7* 87 ± 6* 94 ± 1 74 ± 7 76 ± 8 76 ± 8 78 ± 7 82 ± 6 91 ± 2

25* 0.6 5.7* 3.3* ± ± ± ± 127 2.6 49.0 31.8

80%

26* 0.6 5.2* 4.1* ± ± ± ± 118 2.7 43.5 32.9

60%

23* 0.7 3.8 4.2* ± ± ± ± 96 2.6 37.1 34.7

40%

25 0.7 6.2 4.5* ± ± ± ± 63 2.3 27.7 35.6

20%

7 0.4 2.4 4.6 ± ± ± ± 12 0.8 14.2 31.8 28 0.7 5.6 2.5 ± ± ± ± 129 2.5 52.7 31.6

100%

27 0.7 4.1 2.9 ± ± ± ± 110 2.7 42.7 34.3

80%

27 0.6 4.7 3.8 ± ± ± ± 97 2.6 37.4 36.7

60%

25 0.6 3.2 3.9 ± ± ± ± 80 2.4 33.1 38.1

40%

26 0.7 5.1 3.9 ± ± ± ± 53 2.1 26.1 38.4

20%

4 0.1 3.1 3.4 ± ± ± ±

2

SpO2 (%)

To date, the majority of studies investigating short intermittent exposures to hypoxia, as means of pre-acclimatization to hypoxic environment, have utilized hypobaric hypoxia (Kupper and Schoffl, 2009; Muza et al., 2010) and mainly reported beneficial effects on hypoxic performance (Bartsch et al., 2008; Muza, 2007). The

REST

4.1. Cardio-respiratory responses to hypoxic exercise following SIH

12 0.9 14.7 34.2

The major finding of the present study is that four intermittent exposures to normobaric hypoxia augment V˙ E and SpO2 levels during subsequent high intensity exercise in normobaric hypoxia. These adaptations did not affect either time to exhaustion, or regional blood volume distribution and oxygenation pattern in the exercising limb (vastus lateralis), and accessory respiratory muscles (serratus anterior) during the hypoxic CP test. Even though the tested protocol induced mild hypocapnia during the post CP test, secondary to augmented V˙ E , the regional cerebral oxygenation and blood volume was unaffected.

REST

4. Discussion

SIH group POST

The only significant difference between groups and trials observed was the significantly higher de-oxygenation (↑[HHb]) of the vastus lateralis muscle during the POST compared to the PRE test in SIH group (Fig. 3). Concomitant with the pronounced, significant de-oxygenation (↓[O2 Hb], ↑[HHb]), evident during all tests, was a steady increase in regional blood volume (↑[tHb]). [tHb] was significantly higher during the last part of the test in both groups (80 and 100% endurance time). The significant de-oxygenation pattern (↓[O2 Hb], ↑[HHb]) was similar for the serratus anterior muscle, with the exception that there were no significant differences between PRE and POST trials in neither group (Fig. 4). The serratus anterior regional blood volume was significantly decreased (↓[tHb]) following 20% of relative CP time and remained fairly constant thereafter. The pattern of the response was similar for both groups during both trials (Fig. 4). The changes in oxygen concentration in the cerebral frontal cortex are presented in Fig. 5. The cerebral oxygenation showed a similar trend of significantly decreased oxygenation (↓[O2 Hb], ↑[HHb]) with a tendency, although non-significant, of an increase in regional blood volume (↑ [tHb]). There were no significant differences between groups and testing periods in cerebral oxygenation. Both the de-oxygenation pattern (↓[O2 Hb], ↑[HHb]) and the regional blood volume changes ([tHb]) in all three regions were not significantly different between neither groups nor testing trials.

SIH group PRE

3.4. Regional muscle and cerebral oxygenation during CP

Table 2 Values of the selected cardio-respiratory variables during the PRE and POST constant power test in hypoxia in both SIH and CON group, at the same relative performance time.

and 4th minute of absolute time in SIH group at the POST testing (Table 3). The increased V˙ E was manly driven by an increase in fR and resulted in a significant decline in PETCO2 over both relative and absolute endurance time (Tables 2 and 3). As reported in Table 3 no differences were observed in RPEdys , while the RPEleg values were significantly decreased only in the SIH group at the POST testing.

100%

POST = 338 ± 90 s]; Control [PRE = 350 ± 99 s, POST = 371 ± 106 s]). There was a tendency of an increase in the duration of the CP test in both the SIH (14%) and Control (6%) group. Analysis of the V˙ O2 , V˙ CO2 , HR values, revealed no significant changes between PRE and POST tests in both group. Both, V˙ E and SpO2 were significantly higher during the POST test in the SIH group only. The differences occurred at 40, 60 and 80% of endurance time in V˙ E and at 20, 40, 60 and 100% of endurance time in SpO2 as can be seen in Table 2. Similarly, V˙ E was significantly increased at 3rd

142 ± 17 2.5 ± 0.5 58.2 ± 5.6* 29.1 ± 3.2*

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V˙ E (L) VT (L) fR (breaths min−1 ) PETCO (mmHg)

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Fig. 3. Relative changes from resting values (means ± SE) in the concentration of the oxygenated (A), deoxygenated (B) and total hemoglobin (C) in the vastus lateralis muscle, as measured with NIRS, during the PRE () and POST (䊉) hypoxic constant power tests for the control and short intermittent hypoxic (SIH) exposure groups. Values are plotted as a function of relative exercise time. *Significant difference between PRE and POST values (P < 0.05).

augmented ventilatory response both during rest (Foster et al., 2005; Mahamed and Duffin, 2001) and exercise (Katayama et al., 2001) following SIH protocols, mediated manly by the increased peripheral chemoreceptor sensitivity (Sheel and Macnutt, 2008), has also been well documented (Katayama et al., 2001; Ricart et al., 2000). However, very few studies investigated the effects of normobaric SIH, which can easily be utilized at sea level, on subsequent hypoxic exercise and ventilatory adaptations. Studies have shown the ability of normobaric SIH to induce ventilatory acclimatization as reflected in augmented resting SpO2 (Beidleman et al., 2009; Fulco et al., 2011) with no beneficial effect on subsequent performance in hypobaric hypoxia. Although not significant, a tendency for increased resting SpO2 was also noted in this study, both during the last intermittent exposure and during the rest period of the POST CP test. Interestingly, even though our findings do not

confirm a significant augmentation of resting SpO2 , that has been demonstrated following both normobaric (Beidleman et al., 2009; Katayama et al., 2007) and hypobaric SIH (Beidleman et al., 2008; Rodriguez et al., 2000), the SpO2 was significantly enhanced during subsequent hypoxic exercise. Moreover, the decreased values of RPEleg secondary to increased V˙ E and SpO2 (Table 3) also confirm the beneficial effect of augmented ventilatory exercise response following SIH on leg exertion sensation. Although the RPEleg was significantly decreased during the POST test in the SIH group, a significantly higher de-oxygenation in vastus lateralis was observed (Fig. 3). In contrast to our findings, Katayama et al. (2007) did not show any significant enhancement of maximal and submaximal exercise ventilatory responses following 7 exposures to normobaric hypoxia even though the resting hypoxic ventilatory response was augmented. The possible explanation of the discrepancies might lie in

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Fig. 4. Relative changes from resting values (means ± SE) in the concentration of the oxygenated (A), deoxygenated (B) and total hemoglobin (C) in the serratus anterior muscle, as measured with NIRS, during the PRE () and POST (䊉) hypoxic constant power test for the control and short intermittent hypoxic (SIH) exposure groups. Values are plotted as a function of relative exercise time.

significantly higher hypoxic dose over a short period of time used in our study (4 h × 4 day vs. 1 h × 7 day). Indeed our findings confirm the hypothesis that normobaric SIH can induce exercise ventilatory benefits and are in line with previous studies investigating hypobaric SIH (Katayama et al., 2001; Ricart et al., 2000). Interestingly, Beidleman et al. (2009) argued that the lack of performance benefits following their normobaric SIH protocol cannot be attributed to the different hypoxia mode (i.e. normobaric, hypobaric), but to test timings, since the tests were performed 60 h following the protocol, as compared to previous studies, showing benefits on tests performed within 24 h following hypobaric SIH (Beidleman et al., 2003, 2008). Since a significant individual variability in ventilatory responses to hypoxia has already been shown (Naito et al., 1995) we

also examined within and between subjects results and the interaction between enhanced ventilatory response and augmented performance. As can be seen from Fig. 2, all but two subjects in the SIH group had significantly augmented ventilation during the POST CP test. It is noteworthy, that these two subjects were among the only three subjects that decreased their performance following the SIH. The augmented V˙ E was in all subjects mediated mainly through increased fR whereas the VT did not vary between PRE and POST trials (Tables 2 and 3). Similarly, the SpO2 was also significantly increased in all but two subjects of the SIH group. However, both improved their performance at the POST test even though a 5% decrease in SpO2 was noted. Evidently, the changes in ventilatory responses were much less pronounced in the Control group (Fig. 2). No

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139

Fig. 5. Relative changes from resting values (means ± SE) in the concentration of the oxygenated (A), deoxygenated (B) and total hemoglobin (C) in the left frontal cortex, as measured with NIRS, during the PRE () and POST (䊉) hypoxic constant power test for the control and short intermittent hypoxic (SIH) exposure groups. Values are plotted as a function of relative exercise time.

significant correlation was noted between changes in both SpO2

and V˙ E and change in CP performance time in neither group (r < 0.40). 4.2. Muscle and cerebral regional oxygenation during hypoxic exercise A significant effect of enhanced exercise ventilatory load on respiratory fatigue (Amann et al., 2007; Harms et al., 2000) and a corresponding redistribution of blood flow during heavy exercise, resulting in augmented oxygenation of the respiratory muscles and a reduced oxygenation of the exercising limb muscles, has been demonstrated previously (Legrand et al., 2007). We thus hypothesized that this regional redistribution would also be evident following SIH. As suggested by Harms et al. (2000) higher oxygen flux requirements of the respiratory muscles, at higher ventilation levels, results in a significant decrease in leg muscle oxygenation, most

probably due to a sympathetically mediated vasoconstriction (Harms et al., 1997), which enhances the onset of fatigue. Although they have shown that this phenomenon occurs during heavy load constant power exercise, we did not observe these changes during the hypoxic CP test. Although we considered, that this discrepancies could have been attributed to the differences in training status (highly trained vs. moderately active), the study by Legrand et al. (2007) confirmed that the phenomena is also evident in healthy active subjects. They demonstrated that during last phases of an incremental exercise test, the leg muscle oxygenation decrement was attenuated and related to the accelerated drop in the accessory respiratory muscles oxygenation. This was not observed in our study, since the regional blood volume ([tHb]) in both serratus anterior and vastus lateralis did not show any significant cut-off point, but rather decreased or increased steadily. The observed increased de-oxygenation (↑[HHb]) of the vastus lateralis muscle during the POST testing, did not significantly affect either [tHb] or [O2 Hb]. Moreover, our data also did not show any significant

2

Values are means ± SD. V˙ E , expired ventilation; VT , tidal volume; fR , respiratory frequency, PETCO , end-tidal carbon dioxide partial pressure; SpO2 , arterial oxygen saturation; RPEdys , reported ratings of perceived exertion for

attenuation of the decrease in [O2 Hb] of the vastus lateralis and pronounced decrease in [HHb] of the serratus anterior muscle during the final phase of the exercise. This is in line with the findings of Kowalchuk et al. (2002), who did not observe any changes in leg muscle oxygenation as a result of increased work of breathing during constant power heavy work load exercise. There are a few possible explanations for the observed discrepancies. The lack of redistribution could result from subjects not reaching maximal performance during the exercise test. However, since both the rates of perceived exertions (RPEdys ; RPEleg ) and HR levels during the last phases of the CP test, were comparable to the ones achieved during the final phases of the graded exercise test, not achieving maximal performance is not likely to be a contributing factor. Secondly, even though ventilatory levels were significantly higher during the POST test in the SIH group (∼20%; see Tables 2 and 3), both the accessory respiratory and leg muscle oxygenation was unaffected. It seems reasonable that the extent of the SIH induced increase in ventilation was not sufficient to result in significant blood volume and oxygenation redistribution as shown by Harms et al. (1997). Their intervention increased the respective work of breathing by a greater extent (∼50%), by using a significant external inspiratory resistance during maximal exertion. While this might be a viable explanation for the lack of changes noted in our study, a significant increase in SpO2 (Tables 2 and 3) during POST CP confirmed, that higher ventilatory levels improved arterial oxygenation. This beneficial adaptation could have prevented a possible respiratory muscle oxygenation reduction and thus abolish the blood volume redistribution phenomena. No significant changes were noted in cerebral oxygenation pattern in either group following the protocol (Fig. 5). Since exercise induced hyperventilation has been shown to induce hypocapnia mediated cerebral vasoconstriction that can result in decreased cerebral blood flow in both normoxia (Gonzalez-Alonso et al., 2004) and hypoxia (Imray et al., 2005), we expected that following SIH a decrease in frontal oxygenation would be more pronounced. As shown in Fig. 5 both groups showed a significant de-oxygenation pattern (↓[O2 Hb], ↑[HHb]) throughout the CP tests. However, even though moderate hypocapnia, secondary to significantly increased ventilation was noted during the POST test in the SIH group no significant difference in NIRS oxygenation parameters was noted. Our data are thus in line with other studies showing a significant cerebral de-oxygenation during high intensity exercise in hypoxia (Imray et al., 2005; Subudhi et al., 2007) and moreover show that SIH does not alter the de-oxygenation magnitude. 4.3. Methodological considerations dyspnea; RPEleg , reported ratings of perceived exertion for legs. * Statistically significant changes from PRE (P < 0.05). † Statistically significant difference between SIH and CON groups.

75 ± 7 7.1 ± 1.1 8.2 ± 1.0 77 ± 5 4.6 ± 1.3 5.6 ± 1.2 78 ± 5 3.8 ± 1.5 4.0 ± 1.4 84 ± 3 2.4 ± 1.5 2.5 ± 1.1 92 ± 1 0±0 0±0 75 ± 8 6.7 ± 1.3 8.4 ± 1.3 76 ± 6 5.1 ± 1.3 5.7 ± 0.9 79 ± 6 3.6 ± 2.0 4.1 ± 1.6 84 ± 6 2.3 ± 1.0 2.7 ± 1.0 92 ± 1 0.1 ± 0.2 0±0

2

SpO2 (%) RPEdys RPEleg

104 2.5 42.6 35.4 94 2.5 37.8 37.3 82 2.5 33.2 38.1 50 2.1 23.7 37.9 12 0.5 5.8 5.5 ± ± ± ± ± ± ± ± 109 2.8 40.9 35.8

4

34 0.5 11.1 5.2 50 2.3 21.2 40.1 7 0.3 3.2 1.7 ± ± ± ± REST

11 0.8 13.3 35.5 V˙ E (L) VT (L) fR (breaths min−1 ) PETCO (mmHg)

CON group PRE

1

± ± ± ±

22 0.5 7.7 5.5

85 2.7 31.2 39.9

2

± ± ± ±

29 0.5 9.8 4.5

102 2.7 37.5 37.9

3

± ± ± ±

77 ± 9 4.5 ± 2.1 6.2 ± 1.4 77 ± 9 3.2 ± 1.2 4.6 ± 1.5 82 ± 7 2.0 ± 1.2 3.0 ± 1.2 91 ± 2 0.1 ± 0.3 0±0

2

SpO2 (%) RPEdys RPEleg

28 0.7 5.6 4.5 ± ± ± ± 88 2.5 35.6 37.6 24 0.9 4.7 4.3 ± ± ± ± 55 2.1 26.3 38.3 4 0.1 3.1 3.4 ± ± ± ± 12 0.9 14.7 34.2 V˙ E (L) VT (L) fR (breaths min−1 ) PETCO (mmHg)

16 1.0 14.7 34.4

REST

29 0.5 10.3 5.5

CON group POST

77 ± 10 6.5 ± 1.8 8.3 ± 1.1

94 ± 1 0±0 0±0

1

± ± ± ±

25 0.6 6.6 3.2

87 ± 9 1.8 ± 0.7 2.1 ± 1.1

2

± ± ± ±

30 0.6 9.4 4.2

82 ± 8 2.9 ± 0.7 3.3 ± 1.3*

3

± ± ± ±

33† 0.6 8.8 4.2

80 ± 7 4.3 ± 1.5 4.7 ± 1.5*

4

± ± ± ±

79 ± 9 6.2 ± 0.9 7.8 ± 0.9

± ± ± ± 122 2.7 48.9 31.8

4

22* 0.6 6.2 3.6 ± ± ± ± 112 2.7 42.2 32.9

3

27 0.6 4.8 3.7* ± ± ± ± 96 2.7 37.5 34.6

2

19 0.8 6.1 4.6* ± ± ± ± 62 2.4 27.9 35.4

1

7 0.4 2.4 4.6 ± ± ± ± REST

12 0.8 14.2 31.8 99 2.6 39.9 35.8

± ± ± ±

30 0.7 6.9 4.4

112 2.6 43.4 34.4

± ± ± ±

35 0.7 10.2 4.4

SIH group POST

4 3 2 1 REST

SIH group PRE

Table 3 Values of selected cardio-respiratory variables during the PRE and POST constant power test in hypoxia in SIH and CON group, at the same absolute performance time (min).

36† 0.7 10.8 4.3

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23* 0.6 7.7* 3.9

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One of the main limitations of the previous studies investigating SIH is the lack of a placebo control group, as placebo itself may have potent effects on performance outcomes of different hypoxic interventions (Bonetti and Hopkins, 2009). Indeed, a recent study (Siebenmann et al., 2012), which did not find any significant improvement in performance or related metrics following 28 days of normobaric “live high–train low” protocol (16-h day−1 ) under either 3000 m simulated altitude or strictly controlled placebo normoxia (double blind study design) argued that, a placebo effect could explain the majority of the previous studies findings showing beneficial results without an efficient blinding of the subjects. A matched and sufficiently blinded control group was incorporated in our study design, to exclude the potential placebo effect. The inconsistencies between the studies outcomes could be explained by the purported differences in responses to normobaric and hypobaric hypoxia. Even if a decrease in barometric pressure or a decrease in FIO2 might result in the same absolute decrease in inspired partial O2 pressure an independent effect of barometric pressure per se has been suggested (Conkin

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and Wessel, 2008). Indeed, skepticism exists as to whether the responses to normobaric and hypobaric hypoxia are equivalent and the debate is ongoing (Kayser, 2009; Millet et al., in press). In terms of ventilatory adaptations, significant differences in ventilatory responses to acute hypobaric or normobaric hypoxia have been demonstrated recently (Loeppky et al., 1997; Savourey et al., 2003). Namely, Savourey et al. (2003) showed that subjects exhibit significantly greater hypoxemia, hypocapnia and lower SpO2 levels under hypobaric hypoxia as compared to normobaric hypoxia at the same ambient inspired partial O2 pressure. Accordingly, a limitation of the present study, that normobaric hypoxia was employed for both exposures and exercise testing has to be acknowledged. Hence, our findings cannot be generalized to hypobaric hypoxia (i.e. natural altitude). Even though some studies have reported improvements in hemoglobin, red blood cell content and reticulocyte count following SIH (Rodriguez et al., 1999), our results did not confirm the ability of SIH to induce significant hematological adaptations. Lastly, the SIH protocols in addition to the chronic hypoxic pre-exposures thus provide viable means for inducing beneficial ventilatory responses to hypoxic exercise. Moreover, as shown by Katayama et al. (2005), the subjects who prior to hypoxic exposures undergo a SIH protocol, tend to re-adapt faster even though the time between the two hypoxic exposure may be as long as one month. 5. Conclusion Our results demonstrate that four intermittent normobaric exposures can enhance ventilatory responses to exercise in hypoxia without significantly affecting high intensity endurance performance. The increased exercise ventilation did not result in a significant alteration of the regional cerebral and muscle oxygenation pattern or regional blood volume redistribution from the working leg to the respiratory muscles during hypoxic exercise. Collectively, these data suggest that normobaric SIH can enhance V˙ E and SpO2 during hypoxic exercise, but does not alter regional oxygenation or time to exhaustion. Acknowledgements The study was supported by Grant L7-2413 to Igor B. Mekjavic from the Slovene Research Agency. Tadej Debevec is a recipient of a Young Researcher Scholarship (Slovene Research Agency). We are grateful to Miro Vrhovec for his technical assistance. Appreciation is also extended to all the subjects who participated in the study. References Amann, M., Pegelow, D.F., Jacques, A.J., Dempsey, J.A., 2007. Inspiratory muscle work in acute hypoxia influences locomotor muscle fatigue and exercise performance of healthy humans. Am. J. Physiol. Regul. Integr. Comp. Physiol. 293, R2036–R2045. Bartsch, P., Dehnert, C., Friedmann-Bette, B., Tadibi, V., 2008. Intermittent hypoxia at rest for improvement of athletic performance. Scand. J. Med. Sci. Sports 18 (Suppl. 1), 50–56. Beidleman, B.A., Muza, S.R., Fulco, C.S., Cymerman, A., Ditzler, D.T., Stulz, D., Staab, J.E., Robinson, S.R., Skrinar, G.S., Lewis, S.F., Sawka, M.N., 2003. Intermittent altitude exposures improve muscular performance at 4300 m. J. Appl. Physiol. 95, 1824–1832. Beidleman, B.A., Muza, S.R., Fulco, C.S., Cymerman, A., Sawka, M.N., Lewis, S.F., Skrinar, G.S., 2008. Seven intermittent exposures to altitude improves exercise performance at 4300 m. Med. Sci. Sports Exerc. 40, 141–148. Beidleman, B.A., Muza, S.R., Fulco, C.S., Jones, J.E., Lammi, E., Staab, J.E., Cymerman, A., 2009. Intermittent hypoxic exposure does not improve endurance performance at altitude. Med. Sci. Sports Exerc. 41, 1317–1325. Bonetti, D.L., Hopkins, W.G., 2009. Sea-level exercise performance following adaptation to hypoxia: a meta-analysis. Sports Med. 39, 107–127. Conkin, J., Wessel 3rd, J.H., 2008. Critique of the equivalent air altitude model. Aviat. Space Environ. Med. 79, 975–982.

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