Central chemoreflex ventilatory responses in humans following passive heat acclimation

Respiratory Physiology & Neurobiology 180 (2012) 97–104

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Central chemoreflex ventilatory responses in humans following passive heat acclimation Andrew E. Beaudin, Michael L. Walsh, Matthew D. White ∗ Laboratory for Exercise and Environmental Physiology, Department of Biomedical Physiology and Kinesiology, Simon Fraser University, 8888 University Drive, Burnaby, BC, Canada V5A 1S6

a r t i c l e

i n f o

Article history: Accepted 21 October 2011 Keywords: Carbon dioxide rebreathing Chemosensitivity Control of breathing Heat acclimation Hyperthermia

a b s t r a c t Since there is temperature dependence of pulmonary ventilation (V˙ E ) in response to the normal modulators (i.e. PCO2 /pH, PO2 ), it was asked in this study if passive heat acclimation (HA) modifies the human central chemoreflex ventilatory response to CO2 . Nine males performed normothermic- and hyperthermic modified Read re-breathing tests before and after HA. Heat acclimation consisted of 2 h day−1 exposures to 50◦ C and 20% RH for 10 consecutive days and each exposure elevated rectal temperature to between 38.5 and 39.0◦ C. Ventilatory recruitment thresholds (VRTs) and central chemosensitivity were assessed before and after HA during normothermia with an oesophageal temperature (Tes ) of ∼37◦ C and in hyperthermia when Tes was 38.5–39.0◦ C. Results showed VRT and central chemosensitivities were unaltered by HA (p ≥ 0.375) and hyperthermia increased pre- (p = 0.010) but not post-acclimation (p = 0.332) central chemosensitivity. Additionally, during hyperthermia V˙ E became progressively greater (p = 0.027) relative to corresponding normothermic values in the re-breathing tests. In conclusion, the ventilatory response to hyperoxic CO2 was unaltered by heat Acclimation State. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Thermal hyperpnoea is potentially due to a temperatureinduced increased sensitivity of the peripheral chemoreceptors to CO2 /pH and O2 and the central chemosensitive tissue to CO2 /pH (White, 2006). This follows from evidence demonstrating passively induced hyperthermia augments the ventilatory response to both hypoxia (Curtis et al., 2007; Natalino et al., 1977) and hyperoxic, hypercapnia (Baker et al., 1996; Cunningham et al., 1957; Vejby-Christensen and Strange Petersen, 1973). It remains to be established if this thermal hyperpnoea a result of (1) an increased temperature of the carotid and aortic bodies and the respiratory control area of the ventral-medial surface (VMS) of the medulla oblongata; (2) an increased thermoregulatory drive from the hypothalamus (Boden et al., 2000; See, 1984) directly to the central respiratory control centre; or (3) a combination of these inputs. Changes in temperature of the carotid bodies, while their local environment is maintained at euoxic and eucapnic levels, produces proportional changes of the firing frequencies in the carotid sinus nerves (Gallego et al., 1979; Loyola et al., 1991). As well, in anaesthetized and ventilated cats, changes in the local temperature of

∗ Corresponding author. Tel.: +1 778 782 3344; fax: +1 778 782 3040. E-mail address: [email protected] (M.D. White). 1569-9048/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.resp.2011.10.014

the intermediate area of the VMS of the medulla oblongata, with maintenance of the local chemical environment, gives proportional changes in phrenic nerve firing frequencies (Cherniack et al., 1979). An additional consideration in resolving the mechanism(s) of this response in non-panting animals is that thermal hyperpnoea is still evident after removal of afferent input from central and peripheral chemosensitive tissues to the respiratory control area in the VMS of the medulla oblongata (Loeschcke, 1982; See, 1984). This suggests direct input from hypothalamic thermosensitive tissues (Boden et al., 2000) contributes to the control of thermal hyperpnoea. It follows if there is an input from the hypothalamus (Boden et al., 2000; See, 1984) then thermal hyperpnoea should adapt as do eccrine sweating and cutaneous blood flow thermoregulatory heat loss responses following heat acclimation (HA). Induction of passive hyperthermia over multiple days during HA produces daily hypocapnic, hyperthermic-induced hyperventilation (Beaudin et al., 2009; Cabanac and White, 1995; White, 2006). Irrespective of the cause, 6–24 h of hyperventilation has been shown to give a leftward shift in the V˙ E –PET CO2 relationship and increase the sensitivity of V˙ E to a CO2 stimulus (Brown et al., 1948; Ren and Robbins, 1999). Hence, the purpose of this study was to determine if a passive HA would modify the hyperoxic central chemoreflex ventilatory response to CO2 at either normothermic or hyperthermic Core Temperatures. It was hypothesized HA would lower the ventilatory recruitment threshold (VRT) and increase sensitivity of the hyperoxic central chemoreflex ventilatory response to CO2 .

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2. Methods 2.1. Ethical approval This study was performed according to the Declaration of Helsinki and was approved by the Office of Research Ethics at Simon Fraser University. Each participant was given a 30 min orientation that included an overview of the instrumentation, the protocol and the potential risks. After a 24 h reflection period each participant signed and submitted an informed consent and was instructed they could withdraw from the study at any time without prejudice. 2.2. Participants Nine male participants (mean ± SD; 23.4 ± 3.9 years of age; 1.76 ± 0.09 m; and 70.28 ± 5.75 kg) of normal physique volunteered for the study. The sample size was based upon effect sizes of a 5.0 ± 3.2 mmHg PET CO2 decrease in the VRT and a 0.87 ± 0.4 L min−1 mmHg−1 increase in the sensitivity of the hyperoxic central chemoreflex ventilatory response to CO2 from a normothermic to a hyperthermic Core Temperature (Baker et al., 1996; Sancheti and White, 2006). The sample size that was employed provided a power >0.90. Each participant was a nonsmoker and was asked to avoid consuming caffeine or alcohol, eating and strenuous exercise for a minimum of 4 h prior to each testing session. 2.3. Instrumentation Breath-by-breath measurement of pulmonary ventilation (V˙ E ) and its components were performed with a metabolic cart (Vmax 229c, Sensormedics, Yorba Linda, CA, USA) that collected gas samples at a rate of ∼600 mL min−1 . Sensors and the flowmeter of the metabolic cart were calibrated prior to the start of each testing session as previously outlined (Chu et al., 2007). A two-way mass flow sensor (Sensormedics, Yorba Linda, CA, USA) was connected to a low resistance mouthpiece through which the participant breathed while wearing a nose clip. For CO2 re-breathing tests, a breathing valve controlled by two inflatable balloons was attached to the mass flow sensor which allowed rapid transfer from breathing room air to breathing from a 5-L re-breathing bag (Anesthesia Association Inc., San Marcos, CA, USA) filled with 5.5% CO2 , 33% O2 and balance N2 . Pure medical grade O2 (Praxair, Mississauga, ONT, Canada) was manually titrated into the re-breathing bag via a port in the breathing valve. A paediatric-size thermocouple (9 FR, Mallinckdrot Medical Inc., St. Louis, MO, USA) was used to measure oesophageal temperature (Tes ) during all modified Read re-breathing tests that evaluated the Hyperoxic–Hypercapnic Ventilatory Response (HHCVR) (Casey et al., 1987; Duffin, 2011). The probe was inserted through a nostril into the oesophagus to a depth equivalent to the level of the eighth and ninth thoracic vertebrae (i.e., T8/T9); this position corresponds to the level of the left ventricle (Mekjavic and Rempel, 1990). For the heat acclimation sessions, to give an index of thermal stress, a thermistor (12 FR, Mallinckdrot Medical, St. Louis, MO, USA) was inserted 15 cm past the anal sphincter to measure rectal temperature (Tre ). Skin temperatures (Tsk ) were measured using copper, constantan thermocouples (Omega Engineering Inc., Stanford, CT, USA) positioned on the left temple (Ttemple ), shoulder (Tsh ), lower back (Tlb ) and thigh (Tth ). Temple temperature was expressed on its own and mean Tsk (T¯ sk ) was expressed as the un-weighted mean of Tsh , Tlb and Tth . Thermocouples and thermistors were calibrated across a physiological range of temperatures using a temperature regulated, stirred water bath (Cenco Instruments, Chicao, IL, USA) that was monitored by a platinum thermometer (Fisher Scientific, Nepean, ON, Canada).

Body temperatures were collected simultaneously with each breath via a data acquisition system (National Instruments, Austin, TX, USA) triggered by an analogue flow signal from the metabolic cart and controlled by LabVIEW software (Ver 7.1, National Instruments, Austin, TX, USA) on a personal computer. A portable data logger (Mini Logger Series 2000, Mini-Mitter, Bend, OR, USA) controlled by a personal laptop computer collected Tre at 30 s intervals during all HA sessions. Forehead eccrine sweat rate (E˙ SW ) was measured using a modified version of a ventilated capsule method as previously reported (Beaudin et al., 2009). Also as reported previously (Jay et al., 2007), a laser-Doppler flowmeter 5-in-1 needle probe (MP12-V2, Moor Instruments Ltd., UK) measured cutaneous blood cell velocity of the right temple (CBVTEMPLE ). Arterialized finger capillary blood samples were collected following a 10 min hand immersion in a water bath maintained at 42◦ C (Gaudio and Abramson, 1968). A thin waterproof glove that extended up to the middle of the forearm protected the participant’s limb during the immersion. Samples were analyzed immediately by a portable blood gas analyser (ABL77, Radiometer, Copenhagen, Denmark) for plasma bicarbonate concentration ([HCO3 − ]p ), pH, partial pressures of CO2 (PCO2 ) and O2 (PO2 ), haemoglobin concentration ([Hb]) and haematocrit (Hct). Temperature corrected values of pH, PCO2 , and PO2 are reported for blood samples drawn when the participant was hyperthermic (Andritsch et al., 1981). Alterations in plasma blood volume (PV) with HA were estimated using [Hb] and Hct changes and the equation employed by Dill and Costill (1974). Haemoglobin was measured in g × 100 mL−1 and Hct was expressed as a fraction (volume of RBC (␮L) per blood sample volume (␮L)). Heat acclimation was performed in a walk-in climatic chamber (L – 5.08 m, W – 3.75 m and H – 2.49; Tenney Engineering Inc., Union, NJ, USA). 2.4. Protocol The HA was similar to that of Fox et al. (1963) and included resting exposure to an environment controlled at 50◦ C and 20% RH for 120 min day−1 for 10 consecutive days. For acclimation sessions, each participant was instrumented for Tre while dressed in shorts, T-shirt and running shoes. Next the participant donned gloves, shoes and a hooded vapour barrier suit that was tightly sealed at the waist and ankles. Once dressed, each participant was seated outside the climatic chamber for 5 min in order to acquire a resting Tre . In each 2 h HA session Tre was raised within ∼60 min to between 38.50 and 39.00◦ C where it was maintained by removing portions of the vapour barrier suit or turning on a personal fan. Drinking water was available ad libitum. 2.5. Modified Read re-breathing tests Each participant performed a minimum of five modified Read re-breathing tests (Fig. 1) that evaluated the HHCVR (Casey et al., 1987; Duffin, 2011). The first HHCVR test was for familiarization and was performed with the participant seated in a chair in a comfortable environment of ∼23.0◦ C and ∼30% relative humidity (RH). This test was performed a minimum of one day prior to the next two tests which consisted of one normothermic HHCVR (nHHCVR) and one hyperthermic HHCVR (hHHCVR) test which were separated by a minimum of 45 min. The nHHCVR and hHHCVR tests were performed with the participant immersed up to the shoulders in a water bath maintained at a temperature required to clamp Tes at either a normo- or hyperthermic temperatures (details below). The completion of these three preacclimation HHCVR tests was followed by the 10-day HA protocol

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Fig. 1. Protocol for the pre- and post-acclimation Hyperoxic–Hypercapnic Ventilatory response (HHCVR) tests. The normothermic test (nHHCVR) always preceded the hyperthermic test (hHHCVR). Pre-acclimation tests were followed by a 10 day passive heat acclimation to 50◦ C and 20% RH for 2 h day−1 after which the above protocol was repeated. The only difference between the nHHCVR and hHHCVR was that during the hHHCVR tests, a blood sample was drawn when Tes had stabilized at the hyperthermic level (e.g., BS #3); uREST and iREST represent un-immersed and immersed resting conditioning.

and, subsequently, by post-acclimation nHHCVR and hHHCVR tests. The post-acclimation tests were also performed on the same day, separated by a minimum of 45 min. The pre-acclimation normo- and hyperthermic HHCVR tests were performed 3.4 ± 3.4 days prior to the start of the HA protocol and the two postacclimation tests were performed 1.2 ± 0.4 days following HA. The normothermic HHCVR always preceded the hyperthermic test to prevent any residual effects of a previous hyperthermic state. The mean duration of the nHHCVR and hHHCVR tests was 8.8 ± 0.8 min. For all HHCVR trials, following instrumentation, each participant sat in a comfortable chair for 10 min in a thermoneutral environment (22.7 ± 1.4◦ C and 31% RH) for collection of resting data. Subsequently, the participant wearing only shorts was quickly immersed into the water bath up to the shoulders in less than 1 min. For nHHCVR tests, the mean pre- and post-acclimation water bath temperatures (TH2 O ) were 35.8 ± 0.4◦ C and Tes was held at ∼37.0◦ C. For hHHCVR tests the mean pre- and post-acclimation TH2 O was held at 40.4 ± 0.2◦ C until the Tes reached ∼39.0◦ C. At that time TH2 O was lowered and manually adjusted to ∼38.7 ± 0.2◦ C in order to maintain Tes between 38.5 and 39.0◦ C. Once Tes had stabilized at either the normo- or hyperthermic level for a minimum of 10 min, the participant was asked to voluntarily hyperventilate for 5 min using steady deep breaths to decrease PET CO2 to between 20 and 25 mmHg. After 5 min of hyperventilation and at the end of a full expiration, the participant was switched from breathing room air to breathing from the re-breathing bag containing 5.5% CO2 , 33% O2 , balance N2 . Once switched to the re-breathing bag, the participant took three initial deep breaths to help equilibrate the PCO2 in the bag, lungs and arterial blood with mixed venous blood. Subsequently, the participant returned to normal, relaxed breathing and was instructed to breathe normally for the remainder of the test. The test was terminated when PET CO2 exceeded 60 mmHg or V˙ E surpassed 100 L min−1 . The PO2 within the re-breathing bag was maintained greater than 150 mmHg by manually titrating 100% medical grade O2 into the re-breathing bag. The 5 L re-breathing bag was hidden from sight during all HHCVR tests by an opaque curtain

to prevent visual feedback that might influence the participant’s rate and depth of breathing. Arterialized blood samples were drawn three times (Fig. 1) into capillary tubes during the pre- and post-acclimation HHCVR tests: (1) prior to instrumentation for the nHHCVR; (2) prior to the start of the hHHCVR tests; and (3) during the hHHCVR tests when Tes had stabilized at the hyperthermic level. Additionally, arterialized blood samples were drawn prior to instrumentation on days 1, 2, 4, 6, 8 and 10 of the acclimation protocol. The blood samples drawn on day 1 of the HA protocol were used as the baseline measurement for [Hb] and Hct used in the calculation of PV changes with HA (Dill and Costill, 1974). All 10 acclimation sessions were performed at the same time each day and both the pre- and post-acclimation hHHCVR tests were performed within the same 2 h window at which each participant was acclimated, while the nHHCVR was carried out within the same 2 h period prior to the hHHCVR test. This time control was incorporated as adaptations that arise from a heat acclimation protocol have been shown to be specific to the time of day the acclimation was performed (Shido et al., 1999).

2.6. Analyses Each participant’s VRT for the hyperoxic central chemoreflex ventilatory response to CO2 was determined by plotting breathby-breath data for V˙ E vs. PET CO2 acquired during the modified Read re-breathing tests (Fig. 2). The VRT was defined as the PET CO2 below which CO2 provides no ventilatory stimulation and following which there are proportional increases of V˙ E and PET CO2 (Casey et al., 1987; Duffin, 2011; Ingrassia et al., 1991; Prechter et al., 1990). The VRT was determined using a piecewise linear regression function (Vieth, 1989). Paired, one-tailed t-tests were performed to compare the VRT and sensitivities between the two acclimation conditions (pre vs. post) and Core Temperature conditions as well as the difference within the pre- and post-acclimation normo- and hyperthermic conditions.

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Fig. 2. Breath-by-breath plot of V˙ E vs. PET CO2 in the pre- and post-acclimation hyperthermic hyperoxic–hypercapnic re-breathing tests for one participant. Fitted regression lines through supra-VRT data have been excluded for clarity.

Eccrine sweat rates and CBV on the temple were each plotted as a function of Tes . From these plots Tes onset thresholds were determined using a piecewise linear regression algorithm (Vieth, 1989) written in LabVIEW software (Ver. 7.1, National Instruments, Austin, TX, USA). Additional analysis consisted of a 5 × 2 × 2 repeated measures ANOVA performed on V˙ E , tidal volume (Vt ), breathing frequency (fR ), total breath (TTOT ), inspiratory (TI ) and expiratory times (TE ). Factors included PET CO2 (5 Levels: immersed rest (uREST) or 37.5 mmHg and 40, 45, 50 and 55 mmHg), Core Temperature (2 Levels: normothermic Tes and hyperthermic Tes ) and Acclimation State (2 Levels: pre and post). If there was a significant interaction between PET CO2 with either Acclimation State or Core Temperature, the differences between the pre- and post-acclimation conditions and the normo- and hyperthermic tests were compared used two-tailed paired t-tests. Changes in arterialized blood variables of bicarbonate concentration in plasma ([HCO3 − ]p ), pH, PCO2 and PO2 over the acclimation protocols were analyzed using a oneway repeated measures ANOVA with the factor of Acclimation Day (days 1, 2, 4, 6, 8 and 10). Alpha level was maintained at 0.05 for all comparisons by employing the Bonferonni correction. 3. Results 3.1. Heat acclimation criteria and conditions Successful HA was confirmed by 5 criteria. First, resting un-immersed Tes prior to the post-acclimation nHHCVR

of 37.34 ± 0.19◦ C was significantly lower (p = 0.018) than the un-immersed Tes prior to the pre-acclimation nHHCVR of 37.53 ± 0.13◦ C. Second, on account of the Hct and [Hb] changes, PV increased by 21.69 ± 13.39% (p = 0.011) on the 10th day relative to the 1st day of HA. Third, post-acclimation resting, un-immersed Ttemple of 33.79 ± 1.00◦ C prior to the nHHCVR tended to be lower (p = 0.080) compared to the pre-acclimation Ttemple of 34.34 ± 0.80◦ C. Fourth, the Tes threshold for the onset of E˙ SW significantly decreased from a pre-acclimation 37.61 ± 0.21◦ C to a post-acclimation 37.24 ± 0.24◦ C (p = 0.007). Fifth, the Tes threshold for the onset of temple CBV decreased from a pre-acclimation 37.34 ± 0.12◦ C to a post-acclimation 37.14 ± 0.17◦ C (p = 0.008). The mean Tes maintained during the nHHCVR tests was 37.27 ± 0.12◦ C in the pre-acclimation and 37.12 ± 0.16◦ C in the post-acclimation states. For the pre-acclimation hHHCVR tests, Tes was increased a mean of 1.48 ± 0.18◦ C above un-immersed resting conditions and was clamped at 38.90 ± 0.09◦ C. Within the postacclimation tests, Tes was increased a mean of 1.54 ± 0.23◦ C and stabilized at 38.87 ± 0.07◦ C. Post-acclimation the un-immersed T¯ sk of 32.51 ± 0.76◦ C was not significantly different (p = 0.120) than the un-immersed pre-acclimation T¯ sk of 32.11 ± 0.90◦ C.

3.2. Pre vs. post-acclimation comparisons Fig. 2 shows a representative pre- and post-acclimation plot of V˙ E vs. PET CO2 used to determine the VRT and supra-VRT sensitivities of the hyperoxic central chemoreflex ventilatory response to CO2 . The mean pre-acclimation VRT for the nHHCVR and hHHCVR tests were not significantly different from the post-acclimation nHHCVR (p = 0.555) and hHHCVR (p = 0.901) values, respectively (Table 1). In addition, mean pre-acclimation supra-VRT chemosensitivities were not significantly different than values in the post-acclimation nHHCVR (p = 0.375) and hHHCVR (p = 0.476) tests (Table 1). Pre- and post-acclimation V˙ E during the nHHCVR and the hHHCVR tests were not significantly different at all levels of PET CO2 (F(4,32) = 0.449; p = 0.678; Fig. 3A); that is there was no interaction between PET CO2 and Acclimation State on V˙ E . Acclimation State, irrespective PET CO2 and Core Temperature, had no significant effect on V˙ E , Vt , fR , TTOT , TI and TE (F(4,32) ≤ 1.77; p ≥ 0.204; Fig. 3B–F). Following HA at PET CO2 of 40 mmHg there was a trend for longer TE during nHHCVR relative to hHHCVR (Fig. 3F). There were no significant changes in arterialized blood [HCO3 − ]p , pH, PCO2 or PO2 (F(5,30) ≤ 1.15; p ≥ 0.353) as the HA protocol progressed (Fig. 4A and B). However, there was a significant decrease Fig. 4C in Hct and [Hb] (F(5,30) ≥ 5.67; p ≤ 0.028) by day 6 (p ≤ 0.028) indicative of the observed expansion of plasma volume.

Table 1 Individual and mean (SD) PET CO2 ventilatory recruitment threshold (VRT; mmHg) and supra-VRT chemosensitivity (sensitivity; L min−1 mmHg−1 ) of the pre- and postacclimation hyperoxic central chemoreflex ventilatory response to CO2 at a normo- and hyperthermic Tes performed during a head out immersion. Participant

Pre-acclimation

Post-acclimation

Normothermic

1 2 3 4 5 6 7 8 9 Mean (SD) *

Hyperthermic

Normothermic

Hyperthermic

VRT

Sensitivity

VRT

Sensitivity

VRT

Sensitivity

VRT

Sensitivity

46.5 48.1 46.7 43.2 47.6 48.8 46.7 44.0 47.4 46.6 (1.8)

1.93 2.74 3.91 4.66 2.38 1.11 1.63 7.73 3.50 3.29 (2.02)

47.8 46.1 46.1 43.0 41.6 53.1 50.9 47.3 48.1 47.1 (3.6)

2.06 3.48 4.33 5.49 2.71 2.30 3.26 7.34 3.84 3.87* (1.68)

49.6 46.9 44.0 46.9 46.7 48.8 47.1 46.0 46.9 47.0 (1.6)

1.49 1.86 3.86 6.60 3.71 1.66 1.69 6.76 4.91 3.62 (2.11)

49.6 45.0 47.3 43.8 45.8 51.5 49.3 43.6 49.0 47.2 (2.8)

1.13 2.71 5.98 6.05 3.06 2.43 4.35 7.46 3.51 4.08 (2.05)

Indicates a significant difference between the pre-acclimation normo- and hyperthermic conditions with a p < 0.05.

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* p = 0.05 - pre-acclimation normo- vs. hyperthermic trial. ** p = 0.05 - post-acclimation normo- vs. hyperthermic trial. † p = 0.01 - pre-acclimation normo- vs. hyperthermic trial. †† p = 0.01 - post-acclimation normo- vs. hyperthermic trial. ¥ 0.05 < p = 0.10 - pre-acclimation normo- vs. hyperthermic trial. ¥¥ 0.05 < p = 0.10 - post-acclimation normo- vs. hyperthermic trial.

Fig. 3. Mean pulmonary ventilation (V˙ E ; A), tidal volume (Vt ; B), breathing frequency (fR ; C), total breath time (TTOT ; D), inspiratory time (TI ; E), and expiratory time (TE ; F). Values in each plot were at PET CO2 = 37.5 mmHg for un-immersed rest (uREST) and subsequently when Tes was clamped at either the normo- or hyperthermic level with the volunteer immersed up to the shoulders and while re-breathing CO2 during hyperoxia. Squares are the normothermic pre- (, solid line) and post-acclimation (, dashed line) tests and the circles are the hyperthermic pre- (䊉, solid line) and post-acclimation (, dashed line) tests. Error bars in A represent ±SD, but were excluded from the remaining plots for clarity.

3.3. Pre- and post-acclimation within group comparisons The VRT for the nHHCVR and hHHCVR tests was not significantly (p ≥ 0.627) different within pre- and post-acclimation conditions (Table 1). For the pre-acclimation hHHCVR test, hyperthermia resulted in a significantly greater chemosensitivity (p = 0.010) than that observed during the nHHCVR. In contrast, the mean chemosensitivity during the post-acclimation hHHCVR was not significantly different (p = 0.332) than that observed during the post-acclimation nHHCVR. Core Temperature, irrespective of PET CO2 and Acclimation State, had a significant effect on V˙ E (F(1,8) = 11.75; p = 0.009) its mean being greater during the hHHCVR tests compared to the nHHCVR tests (Fig. 3A). In addition, the interaction between PET CO2 and Core Temperature was significant for V˙ E (F(4,32) = 7.13; p = 0.004). That is, irrespective of Acclimation State, V˙ E was significantly higher at

all levels of PET CO2 during the hHHCVR compared to the nHHCVR. The difference in V˙ E between the normo- and hyperthermic tests also became larger at higher levels of PET CO2 . There was no significant interaction between PET CO2 and Acclimation State for either Vt (F(4,32) = 1.89; p = 0.157) or fR (F(4,32) = 0.431; p = 0.677) with both being similar during the nHHCVR and hHHCVR pre- and postacclimation tests at the different levels of PET CO2 (Fig. 3B and C). The interaction between PET CO2 and Core Temperature, irrespective of Acclimation State, was significant (F(4,32) = 3.58; p = 0.039) for Vt . Tidal volume became larger during the hHHCVR tests as PET CO2 increased, but there was no significant interaction between PET CO2 and Core Temperature for fR (F(4,32) = 2.177; p = 0.118). Irrespective of both Acclimation State and Core Temperature, PET CO2 had a significant effect on TTOT (F(4,32) = 5.43; p = 0.015), TI (F(4,32) = 4.28; p = 0.031) and TE (F(4,32) = 10.26; p = 0.003) (Fig. 3D–F). Total breath time (Fig. 3D) and TE (Fig. 3F) initially increased from

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[HCO3- ]p (mmoL L-1)

28

7.48

26

.

37.5 mmHg and then decreased with the progressive increases in PET CO2 . In contrast, TI initially decreased from 37.5 mmHg and then increased proportionately to PET CO2 (Fig. 3E). Core Temperature did not influence TTOT (F(4,32) = 1.29; p = 0.290), TI (F(4,32) = 0.003; p = 0.955) or TE (F(4,32) = 2.14; p = 0.182) responses across the different levels of PET CO2 . At all sampling points, mean values for [HCO3 − ]p , pH, PCO2 and PO2 were not influenced by HA (Table 2). The pre- and postacclimation mean [HCO3 − ]p measured during the hHHCVR tests when Tes was clamped at the hyperthermic level were not different (p = 0.264) from the normothermic – un-immersed value measured prior to the start of the hHHCVR tests. For pH there was a trend (0.069 ≤ p ≤ 0.098) to be more alkaline during both the pre- and post-acclimation hHHCVR when Tes was clamped at the hyperthermic level compared to normothermic – un-immersed samples drawn prior to starting the hyperthermic tests. The PCO2 during the pre-acclimation tests also showed a trend (p = 0.081) to be lower and PO2 was significantly higher (p ≤ 0.016) when Tes was clamped at the hyperthermic level during the pre-acclimation tests (Table 3).

7.52

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24

7.40 22 7.36 20 7.32

90

55

80

50

70

45

60

40

50

35

40

PO2 (mm Hg)

60

0.52

4. Discussion There were two principal findings in the present study. First, neither the VRT nor the supra-VRT chemosensitivity of the hyperoxic central chemoreflex ventilatory response to CO2 at either a normoor hyperthermic Core Temperature were modified by a 10-day passive heat acclimation. Second, Acclimation State had no effect on pulmonary ventilation in either the normo- or hyperthermic conditions over the entire range of PET CO2 from 37.5 to 55 mmHg. Similar to the VRT and supra-VRT chemosensitivity, the breathing pattern was not modified by HA during the ventilatory responses tests to CO2 (Fig. 3). The positive interaction between PET CO2 and Core Temperature for V˙ E (F = 7.13, p = 0.004) and for Vt (F = 3.58, p = 0.039) was uninfluenced by HA. This confirms that temperature and hypercapnia interacted, giving progressively greater responses acutely, but that these ventilatory responses were unmodified by repeated heat exposure. Passive HA did cause an adaptation of exercise V˙ E

16.5

C

Hct: Day 6 vs. 1 - p = 0.026 Hct: Day 8 vs. 1 - p = 0.028 Hct: Day 10 vs. 1 - p = 0.009

0.50

16.0 -1

Hct (% Fraction)

100

B

[Hb] (g dL )

PCO2 (mm Hg)

65

0.48

15.5

0.46

15.0

0.44 0.42

14.5

[Hb]: Day 6 vs. 1 - p = 0.027 [Hb]: Day 8 vs. 1 - p = 0.031 [Hb]: Day 10 vs. 1 - p = 0.008

1 2

4

6

.

14.0 8

10

Acclimation Days Fig. 4. Normothermic, resting arterialized blood plasma bicarbonate concentration ([HCO3 − ]p ; 䊉) and pH (; A), partial pressure of CO2 (PCO2 ; ) and O2 (PO2 ; ×; B), haematocrit (Hct; ) and haemoglobin concentration ([Hb]; ; C) measured on the 2nd, 4th, 6th, 8th and 10th day of acclimation prior to instrumentation. Error bars represent ±SD and have been omitted from C for clarity. Only significant p-values are shown for the comparisons between baseline measures on day 1 and Hct as well as [Hb] values on days 2, 4, 6, 8 and 10 of HA. Mean [HCO3 − ]p , PCO2 and PO2 are for n = 7 and the Hct, [Hb] are for n = 6.

(Beaudin et al., 2009) suggesting an adaptation of thermoregulatory input to breathing from cutaneous (Wenger, 2008) and/or pre-optic anterior hypothalamic (Boden et al., 2000; Christman and Gisolfi, 1985) temperature sensitive tissues. In addition to these potential afferent inputs (Boden et al., 2000; Christman and Gisolfi, 1985; Wenger, 2008), another possible influence on V˙ E in the hyperthermic condition are group III and IV afferents in skeletal muscle as they have been shown to increase their firing rates due to increases in their temperature (Hertel et al., 1976) and these are thought to stimulate breathing. The mean pre- and post-acclimation nHHCVR and hHHCVR VRT of ∼47 mmHg within the present study are similar to

Table 2 Pre- and post-acclimation plasma bicarbonate concentration ([HCO3 − ]p ), pH, PCO2 and PO2 from arterialized blood samples (n = 6). Normothermic – un-immersed samples were taken prior to instrumentation for both the n- and hHHCVR tests, prior to immersion for the hHHCVR test and again during immersion and prior to starting the modified Read re-breathing when Tes was clamped at the hyperthermic level. Variable

[HCO3 − ]p (mmol L−1 ) pH PCO2 (mmHg) PO2 (mmHg) * ** ***

Pre-acclimation

Post-acclimation

nHHCVR

hHHCVR

nHHCVR

hHHCVR

Normo – un-immersed

Normo – un-immersed

Hyper – immersed

Normo – un-immersed

Normo – un-immersed

Hyper – immersed

25.48 (2.02) 7.39 (0.03) 44.83 (3.76) 84.17 (9.35)

25.57 (2.44) 7.36 (0.02) 46.33 (5.54) 75.17*** (7.25)

24.03 (2.39) 7.42*** (0.06) 38.50*** (7.56) 85.17* (4.83)

25.68 (1.45) 7.38 (0.03) 45.50 (3.27) 82.67 (8.04)

25.40 (1.14) 7.37 (0.02) 45.83 (3.06) 74.50** (5.96)

25.17 (1.50) 7.41*** (0.05) 41.83 (6.88) 86.67* (4.93)

Significant difference between the normo – un-immersed and the hyper – immersed conditions within the hHHCVR tests with a p-value ≤0.05. Significant difference in the normo – un-immersed values between the post-acclimation nHHCVR and the hHHCVR with a p-value ≤0.05. Trend for a difference between normo – un-immersed and the hyper – immersed values during the hHHCVR tests with a 0.05 < p ≤ 0.10.

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103

Table 3 Mean resting immersed (iREST) responses of PET CO2 (n = 9) to an increase in Tes from a normo- to hyperthermic value. Pre-acclimation

Post-acclimation

Normothermic PET CO2 (mmHg) Mean SD †

39.40 2.70

Hyperthermic PET CO2 (mmHg) 34.26† 8.53

Normothermic PET CO2 (mmHg) 40.55 2.28

Hyperthermic PET CO2 (mmHg) 33.92† 5.04

Significant difference between the normothermic and the hyperthermic conditions in both pre- and post-acclimation conditions with a p-value ≤ 0.01.

the normo- and hyperthermic VRT of ∼48 mmHg observed by Baker et al. (1996), ∼45.3 mmHg observed by Duffin et al. (2000) and ∼49 mmHg observed by Jensen et al. (2005). It is important to note that each of these prior studies employed a similar hyperoxic–hypercapnic modified Read re-breathing protocol. The supra-VRT chemosensitivity range for the nHHCVR tests of 1.11–7.73 L min−1 mmHg−1 observed in the present study, even though higher than the normothermic range of ∼1.50–2.72 L min−1 mmHg−1 observed by Baker et al. (1996), and the 1.0–5.95 L min−1 mmHg−1 range observed by Hirshman et al. (1975), are similar to more recent studies where a mean of ∼4.5 L min−1 mmHg−1 was observed (Jensen et al., 2005). The range of the hHHCVR supra-VRT chemosensitivity within the present study of 2.06–7.46 L min−1 mmHg−1 is larger than that of 2.43–3.27 L min−1 mmHg−1 observed by Baker et al. (1996) who employed a similar protocol, but these differences are the likely result of the large–inter-individual variability of HHCVR responses (Hirshman et al., 1975). The HA of the volunteers was established on five criteria as described in the literature (Armstrong and Maresh, 1991; Wenger, 2008). Significant responses for each of the reductions in resting Core Temperature, increases of plasma volume, as well as reductions in Tes thresholds for the onset of eccrine sweating and increases in CBVTEMPLE gave support that HA was successfully achieved in this study. In addition, there was a trend for a reduction in temple skin temperature that also supported HA of the volunteers. An exhaustive literature review did not reveal any previous studies that explored the effect of HA on peripheral or central chemosensitive tissues to a hyperoxic, hypercapnic stimulus in either a human or animal model. Krivoshchekov and Divert (1997) explored changes in human chemosensitivity following acclimation to a cold environment and contrary to the present findings they report an increase in the hypoxic and hypercapnic ventilatory responses following 10 days of exposure to 13◦ C for 2 h day−1 . It is difficult to compare the present results to those of Krivoshchekov and Divert (1997) as acclimation to a cold environment produces very different physiological adaptations than those occurring with acclimation to a hot environment. Additionally, unlike heat acclimation where thermoregulatory adaptations are generally consistent between individuals, cold acclimation can take several different forms (i.e., metabolic, insulative or hypothermic) and may vary between individuals depending on the cold acclimation protocol employed (Bittel, 1992). It is important to note that the assessment of the effect of HA on chemosensitivity was limited to the range of PET CO2 above the VRT (i.e., from ∼47 to 55 mmHg; Table 1). On the other hand, over a larger range of PET CO2 from 37.5 to 55 mmHg, there was a positive interaction between PET CO2 and Core Temperature for both VE and Vt . These positive interactions demonstrate an enhancing effect of Core Temperature on CO2 central chemosensitivity over a larger physiological range of PET CO2 than that employed in the assessment of the affect of HA on supra-VRT chemosensitivity. This suggests future studies of potential HA effects on central chemosensitivity during CO2 re-breathing tests should consider pulmonary ventilation responses across the entire physiological

range of PET CO2 . An additional potential area for future research is of the effect of HA on the timing of breathing. The TE at a PET CO2 of 40 mmHg showed a trend for a reduction following HA (Fig. 3F) supporting that a change in the pattern of breathing may be adopted after repeated heat exposures. For pH, PCO2 , and [Hb], arterialized fingertip blood samples have been shown to be a reliable substitute for arterial punctures with approximate mean biases of +1 mmHg for PCO2 , +0.01 for pH and −0.18 g dL−1 for [Hb] (Zavorsky et al., 2005, 2007). Arterialized fingertip blood samples have been shown to underestimate arterial PO2 by ∼10 mmHg (Zavorsky et al., 2007). Finally, [HCO3 − ]p in arterialized blood samples has been observed to be similar to arterial values (Linderman et al., 1990), but no literature was uncovered comparing Hct in arterialized blood samples to arterial blood samples. Therefore, even though the current mean [HCO3 − ]p , pH, and PCO2 appeared to be high and PO2 values appeared to be low relative to typical arterial values, they are similar to previously published results using the same fingertip sampling site and method. Limitations of the present study include the possibility there was some apprehension and anxiety due to the prior hyperventilation and/or unfamiliarity with breathing from a mouthpiece and this may have altered the observed VRT and sensitivities (Jensen et al., 2005). These were not considered to have influenced the ventilatory responses observed as each participant had performed a minimum of one familiarization modified Read re-breathing test prior to the pre-acclimation tests and each had prior experience breathing from a mouthpiece. It is also possible the passive heat acclimation protocol may not have provided sufficient thermal and/or ventilatory strain to produce an adaptation of the hyperoxic central chemoreflex ventilatory response to CO2 . It is not clear if the 2 h day−1 increase in Tes was an adequate degree of thermal stimulation to chronically alter the response of the central chemosensitive areas to an increase in temperature as, to the best of our knowledge, this has never been explored. Then again, chronic euoxic-hyperventilation in normothermic humans has been shown to lower the VRT and increase the sensitivity to CO2 (Brown et al., 1948; Ren and Robbins, 1999). This reported decrease in the VRT and increased sensitivity (Brown et al., 1948; Ren and Robbins, 1999) was the likely product of decreased arterial [HCO3 − ] and increased arterial pH accompanying chronic euoxic-hyperventilation (Dempsey et al., 1975). Hyperthermia is known to produce a hyperventilation in humans (White, 2006). The repetitive hyperthermic-hyperventilation during the 2 h acclimation sessions when Tre was increased and held constant between 38.50 and 39.00◦ C is believed to be a sufficient stimulus to produce similar changes in arterialized [HCO3 − ]p and pH during resting conditions as seen with chronic euoxic-hyperventilation. However, normothermic arterialized plasma [HCO3 − ] and pH, as well as PCO2 and PO2 were not altered following the HA protocol. In conclusion, this study showed that the normo- and hyperthermic VRT and chemosensitivity to hyperoxic CO2 , along with the breathing pattern of the central chemoreflex ventilatory response were not altered by a 10 day passive heat acclimation protocol utilizing controlled hyperthermia.

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