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Intermittent hypoxia and the chemoreflex control of breathing Koehle, Michael Stephen 2006

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INTERMITTENT HYPOXIA AND THE CHEMOREFLEX CONTROL OF BREATHING by MICHAEL STEPHEN KOEHLE B.Sc.H., Queen's University at Kingston, 1993 M.Sc, The University of Toronto, 1995 M.D., The University of Toronto, 1999 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Human Kinetics) THE UNIVERSITY OF BRITISH COLUMBIA August 2006 © Michael Stephen Koehle, 2006 ABSTRACT Rationale: Intermittent Hypoxia (IH) consists of bouts of hypoxic exposure interspersed with normoxic intervals. In animals, there is some evidence that multiple brief short duration exposures to intermittent hypoxia (SDIH) provoke more profound changes in chemosensitivity than longer duration bouts of intermittent hypoxia (LDIH). The purpose of this study was to test the hypothesis that SDIH would have differential effects from LDIH on chemosensitivity during rest and exercise in humans. Methods: Ten males underwent two intermittent hypoxic protocols of 7 days duration/each. The LDIH protocol consisted of daily 60-minute exposures to normobaric 12% O2. The SDIH protocol consisted of twelve 5-minute bouts of normobaric 12% O2, separated by 5-minute bouts of normoxia. Measured resting variables included the hypoxic ventilatory response (HVR), hypercapnic ventilatory response (HCVR), C02 threshold and CO2 sensitivity. Submaximal exercise variables included minute ventilation, oxygen saturation, hyperoxic and hypercapnic ventilatory response in both hypoxia and normoxia. Peak exercise variables included power and oxygen consumption in hypoxia. Measurements were made immediately prior to intermittent hypoxic training and on the first day following IH. Resting measures were repeated 7 days following IH. Results: For both protocols, the HVR was significantly (p< 0.05) increased after IH. One week post IH, the HVR was not different from pre-IH. The HCVR was increased and remained elevated at 7 days post-IH (p<0.01). The CO2 sensitivity was unchanged by either intervention. In hypoxia and hyperoxia, the CO2 threshold was significantly reduced following IH (p<0.05). The submaximal minute ventilation, hyperoxic and hypercapnic responses in normoxia and hypoxia were unchanged by IH. Submaximal oxygen saturation and peak power were both increased (p<0.05), while maximal ventilation and oxygen consumption were unaltered. There were no significant differences between the two IH protocols for any of the above measures. Conclusions; A 7-day IH protocol causes increases in the HVR and HCVR at rest and a left-shift in the CO2 threshold and an improvement in oxygen saturation during submaximal hypoxic exercise. SDIH is no more efficacious than LDIH at effecting these changes in respiratory control. ii TABLE OF CONTENTS Abstract • ii Table of Contents iiList of Tables vList of Figures viii Co-authorship Statement x Chapter 1 - Introduction 1 Proposed Mechanism of Intermittent Hypoxia.. 2 Duration of Intermittent Hypoxia 8 Hypobaric vs. Normobaric Intermittent Hypoxia 8 Poikilocapnia vs. IsocapniaHypoxia Duty Cycle 9 Responses to Carbon Dioxide and Intermittent Hypoxia 10 Respiratory Drive during Exercise 12 Summary 14 Research Questions 15 Hypotheses 7 Purpose 1References 8 Chapter 2 - Repeated Measurements of Hypoxic Ventilatory Response as an Intermittent Hypoxic Stimulus 23 Research Question 4 Methods 2Data and Statistical Analysis 26 iii Results 26 Discussion 32 Methodological Considerations. 34 Conclusions 35 References 6 Chapter 3 - Intermittent Hypoxia and its Effect on Resting Measures of Chemoresponse 8 Research Questions 39 Methods 3Hypoxic Ventilatory Response 42 Hypercapnic Ventilatory Response 4 Modified Rebreathing 45 Intermittent Hypoxic Training 47 Follow-up 48 Statistics 9 ResultsHypoxic Ventilatory Response 50 Modified Rebreathing 54 Hypercapnic Ventilatory Response 6 Associations between Chemosensitivity Measures 57 Discussion 58 Hypoxic Ventilatory Response 5Hypercapnic Ventilatory Response 9 Modified Rebreathing 5SDIH vs. LDIH 63 Limitations 5 Conclusions 6 References 67 iv Chapter 4 - Intermittent Hypoxia and its Effect on Exercise Chemosensitivity...69 Research Questions • • .70 Methods 7Exercise Test 71 Intermittent Hypoxia Protocol 74 Follow-upData Analysis 5 Results 7Submaximal Exercise Test 75 Hypoxic Graded Exercise Test 8 Discussion 81 Minute Ventilation During Normoxic Exercise 81 Minute Ventilation During Hypoxic Exercise 3 Hyperoxic Test 85 Hypercapnic Test 7 Oximetry during Submaximal Exercise 88 Maximal Exercise Test 89 SDIH vs. LDIH 90 Limitations 1 Conclusions 2 References. 93 Chapter 5 - Conclusions 6 References 100 Appendix I - Certificates of Ethical Review 103 Appendix II - Informed Consent Forms 107 Appendix III - Physical Activity Readiness Questionnaire 116 Appendix IV- Data Tables 118 LIST OF TABLES 1.1: Summary of selected research on intermittent hypoxic training and ventilatory control in humans 5-7 2.1: Anthropometric and spirometry data for all subjects 27 2.2: Resting respiratory parameters and hypoxic ventilatory response (HVR)...28 3.1: Anthropometric, Respiratory and Exercise Baseline Data 50 4.1: Exercise Minute Ventilation Pre- and Post-Intermittent Hypoxia (IH) 75 4.2: Mean decreases in Minute Ventilation following 3 breaths of hyperoxia (Pre-and Post-IH) 76 4.3: Mean increases in Minute Ventilation following 1 breath of hypercapnia (Pre-and Post-IH) 7 6.1: HVR Results during the SDIH protocol.... 119 6.2: HVR Results during the LDIH protocol 120 6.3: HCVR Results during the LDIH and SDIH protocols 121 6.4: Hypoxic modified rebreathing method results during the LDIH and SDIH protocols 122 6.5: Hyperoxic modified rebreathing method results during the LDIH and SDIH protocols .123 6.6: Submaximal minute ventilation values during exercise before and after SDIH , 124 6.7: Submaximal minute ventilation values during exercise before and after LDIH 125 6.8: Submaximal saturation values during hypoxic exercise 126 vi 6.9: Hyperoxic Test ventilation values during normoxic exercise before and after SDIH 127 6.10: Hyperoxic Test ventilation values during normoxic exercise before and after LDIH '. 128 6.11: Hypercapnic Test ventilation values during normoxic exercise before and after SDIH .....129 6.12: Hypercapnic Test ventilation values during normoxic exercise before and after LDIH .....130 6.13: Hyperoxic Test ventilation values during hypoxic exercise before and after SDIH 131 6.14: Hyperoxic Test ventilation values during hypoxic exercise before and after LDIH 2 6.15: Hypercapnic Test ventilation values during hypoxic exercise before and after SDIH 133 6.16: Hypercapnic Test ventilation values during hypoxic exercise before and after LDIH 4 6.17: Peak Ramp time, oxygen consumption V02 and ventilation during a graded exercise test in hypoxia and after SDIH 135 6.18: Peak Ramp time, oxygen consumption V02 and ventilation during a graded exercise test in hypoxia and after LDIH 136 vii LIST OF FIGURES 2.1: Sample data from one HVR test on one subject 29 2.2: HVR plot using the data from Figure 2.1 31 2.3: Summary plot of all HVR results for all subjects 32 3.1 Study Paradigm 41 3.2 Sample data from an individual modified rebreathing test 47 3.3: Mean (±SD) Hypoxic Ventilatory Response (HVR) vs. Time 51 3.4: Individual Hypoxic Ventilatory Response (HVR) vs. Time during the LDIH Protocol. Thick black line denotes mean response 52 3.5: Individual Hypoxic Ventilatory Response (HVR) vs. Time during the SDIH Protocol. Thick black line denotes mean response 53 3.6: Mean (+SD) Hypoxic Ventilatory Response (HVR) vs. Time by Protocol Order.. 53.7: Mean (±SD) Carbon dioxide Threshold in Hyperoxia vs. Time 55 3.8: Mean (±SD) Carbon dioxide Threshold in Hypoxia vs. Time 55 3.9: Mean (±SD) Hypercapnic Ventilatory Response (HCVR) vs. Time 57 4.1 Exercise Testing Protocol.... 72 4.2: Mean (±SD) Oxygen Saturation (%) during Submaximal Hypoxic Exercise vs. Intermittent Hypoxia Protocol 78 viii 4.3: Mean (±SD) Peak Wattage vs. Intermittent Hypoxia Protocol 79 4.4: Mean (±SD) Peak Oxygen Consumption (L#min"1) vs. Intermittent Hypoxia Protocol 80 4.5: Mean (±SD) Peak Exercise Ventilation (L»min~1) vs. Intermittent Hypoxia Protocolix CO-AUTHORSHIP STATEMENT A version of Chapter 2 has been previously published as; Koehle MS, Foster GE, McKenzie DC and Sheel AW (2005) Repeated measurement of hypoxic ventilatory response as an intermittent hypoxic stimulus Resp Physiol Neurobiol 145(1): 33-39. MS Koehle was the primary author and played the principal role in identification and design of the research programme, performance of research, data analysis and manuscript preparation. GE Foster assisted in the performance of research. DC McKenzie assisted in identification and design of the research programme and manuscript preparation. AW Sheel assisted in identification and design of the research programme, data analysis and manuscript preparation. CHAPTER 1: INTRODUCTION The term Intermittent Hypoxia (IH) refers to an exposure to multiple brief bouts of hypoxia over a period of time. This length of time can be as short as a few minutes or as long as several weeks. IH has been shown to increase red cell mass1, blood pressure2 and cerebrovascular and sympathetic responses to hypoxia3 4. IH can cause alterations in the chemoreflex control of breathing in humans that may have potential clinical or ergogenic applications. There is some evidence that it may protect the heart from ischaemia and have the potential to improve ventilation in spinal cord transection patients6. For the respiratory system, IH can increase an individual's ventilatory response to hypoxia at rest7"11. There is some evidence that IH can also increase exercise ventilation during exercise tests at simulated altitude (4500 metres)12. This increased ventilation is associated with improved arterial oxygen saturation during exercise. IH does not appear to increase exercise ventilation at sea level13. However, although no research has examined exercise ventilation at sea level and simulated altitude following the same IH exposure. Proposed Mechanism of Intermittent Hypoxia Due to its invasive nature, the majority of the research into the mechanism of IH has been performed in animal models. These concepts are applied to human physiology with appropriate caution. Acute hypoxic exposure leads to a series of alterations in neural control of respiration; these are the short-term hypoxic phrenic response, the post-hypoxia frequency decline and phrenic Long-term Facilitation (LTF). The short-term hypoxic phrenic response is an increase 2 in integrated phrenic amplitude during the hypoxic exposure. This modulation does not persist beyond the hypoxic exposure, but it can be augmented by intermittent hypoxia through a serotonin-mediated mechanism14. Following the hypoxic exposure, a transient decrease in phrenic motorneuron frequency occurs that returns to baseline after several minutes.14 Subsequent to the hypoxic exposure, there is a persistent increase in phrenic motorneuron output amplitude lasting minutes to hours, termed the phrenic LTF15. LTF following an acute hypoxic exposure is a form of neural "plasticity". Plasticity is defined as "a persistent change in the neural control system based on prior experience"16. With repeated exposures to hypoxia (intermittent hypoxia) this increased phrenic motorneuron output (LTF) is further augmented14. This amplification of the LTF is believed to be a form of "metaplasticity" whereby a prior exposure (to hypoxia) modifies one's ability to express plasticity16. This metaplasticity can be blocked using the serotonin blocker methysergide14, indicating that the mechanism is at least partially serotonin-mediated. Other mechanisms may also play a role in the generation of LTF, such as the nitric oxide pathway15. Following intermittent hypoxia, the augmented LTF can be induced with either hypoxia or electrical carotid sinus nerve stimulation, indicating that this plasticity occurs (at least partially) through central facilitation of chemoreceptor afferents as opposed to occurring entirely at the carotid peripheral chemoreceptors themselves14. LTF can occur in response to hypoxia in carotid-denervated cats, indicating that there may be a possible role for the direct effect of hypoxia on the CNS neurons themselves.17 3 Duration of Intermittent Hypoxia Many different IH protocols have been utilized in previous studies in humans (see Table 1.1). Exposures have ranged from twenty minutes to two hours in length, and have typically been repeated between 7 and 14 times. The optimal IH intervention for increasing hypoxic ventilatory response is unknown. One way to better characterize the minimum duration of IH on ventilation would be to measure hypoxic ventilatory response (HVR) daily during the administration of an IH regime. When working with hypoxia and hypoxic sensitivity, one must be careful to ensure that the measurements used in a study are not interventions themselves. During a single HVR test, a subject receives a short exposure to significant hypoxia (lasting approximately 5 minutes) where F|02 (fraction of inspired oxygen) can reach as low as 5%. An HVR test therefore represents a shorter but more severe hypoxic exposure than the typical bout of hypoxia used during IH. It is possible that the brief, more profound exposures of an HVR test, if repeated daily, could present an intermittent hypoxic stimulus and therefore affect ventilation during subsequent hypoxic exposures. Before incorporating daily assessment of HVR into a study, the significance of this potential co-intervention would need to be assessed. 4 Paper N Intervention Testing HVR HCVR HCVR sb Modified Read Rebreathe V02max Exercise Ventilation Haem. Comments Foster et al. 200618 9JSDIH 9c?LDIH Normobaric 12%02 10 of 12 days SDIH/LDIH -V02max, min Ve, Fb at various %max N/A N/A N/A N/A -OA (at sea level) -0A (at sea level) N/A -isocapnic Townsend et al.20058 12c?A 11c?B 10c?C Normobaric 16.3 %02 A=20dX8-10h B=4 X (5d X 8-10h) C=Control -HVR (pre-, Post-) -submaximal exercise Ve (Pre-, after 4,10 19 nights) -increased in both IH groups N/A N/A N/A N/A -increased after 4 nights of hypoxia for both IH groups N/A -exercise ventilation measured in normoxia -change in exercise ventilation correlated to change in HVR Foster et al. 20057 9<?SDIH 961DIH Normobaric 12% 02 10 of 12 days SDIH/LDIH -HVR days 1,3,5,8,10,12 increased in both groups -0A N/A N/A N/A N/A N/A -isocapnic Katayama etal. 20051' 7c?A-E 7<?A-C 8<3B-E 7c?B-C Normobaric 12.3% 02 A = 3h X 7d B =3h X 14d -HVR, pre-,post-, 1-2-wks post--HCVR, pre-,post-, 2-wks post--increased in both groups, back to N w/l 2/52 -incr. only after 2/52 of IH N/A N/A N/A N/A N/A N/A Katayama etal. 200420 8c?E 7c?C Normobaric 12.3% 02 3h X 14d -maximal, submaximal X 2, TT -pre- and post -haematology N/A N/A N/A N/As -0A in V02max -| sub max V02 (efficiency?) -0A in Hb, Hct, RBC, Ret -trend to improved TT performance Ainslie et al. 200321 12<? Normobaric 4300mX8-9hX5d -HVR, HCVR pre test X 2, post, and 5d-post -increased by 1.6L/min/% Sa02 increase in slope -left shift in intercept N/A N/A N/A N/A N/A -HVR used PET02 . Hendrikse n and Meeuwsen 200322 12<? Hvoobaric 2hX2500mX10d -2h cycling 65%HRR -crossover control -V02max, Wingate, Hb, Hct -pre-, 9d post-N/A N/A N/A N/A -0A in V02max fanaero bic power N/A -THb, Hct -triathletes Table 1.1 Summary of Selected Research on intermittent hypoxia and ventilatory control on humans Paper N Intervention Testing HVR HCVR HCVR Sb Modified Read Rebreathe V02max Exercise Ventilation Haem. Comments Mateika et at. 2003" 7cJ Normobaric 4'X 8%02 X 8 -MRR test pre- and 1h post--PO2=50 and 140 N/A N/A N/A -increase in slope -0A threshold N/A N/A N/A Katayama etal. 200313 6c?C 6c?E HvDobaric (4500m) -90' X 9 (over 3/52) -3k run time, run to exhaustion, V02max, haematology -pre-, post, 3/52 post-N/A N/A N/A N/A -0A in V02max -0A (at sea level) -1 submax VOi (efficiency?) -0A in Hb, Hct, RBC, Ret, Epo, Ferritin -|3k times, submax V02 -f run time to exhaustion -trained runners Fahlman etal. 200224 5c? 7? Normobaric -novel repeat HVR circuit -2'X8.3%02X4, alternating w/ 21%02 -repeated HVR measurements following square 4m waveform -'steady' response -variable response N/A N/A N/A N/A N/A N/A -CVwas70% Katayama etal. 20029 8c?E 6c?C Hvoobaric (4500m) •60'X7d -HVR, HCVRsb, pre- post--Ve @ 40, 70, 100%max pre-,post -fHVR at rest - 0A in C N/A -non-signrR cant-t post-N/A N/A - 0A at any exercise level N/A -exercise at sea level Katayama etal. 200112 6c? HvDobaric (4500m) -60' X 7d -HVR, HCVR, HCVRsb, max and submax Ve, VO2 -pre-, post-, 7d post--|HVR post-and7d post--incr by -90% -0A -T post--0A 7d post-N/A -0Ain V02max -| Ve/V02 and Sa02 @ rest, 40%, 70% of max -t Sa02 @ max N/A -maximal and submaximal tests af 4500m Katayama etal. 20012 14c? HvDobaric (4500m) -60'X7d -ventilatory and CV responses to hypoxia -pre-, post-N/A N/A N/A N/A N/A - 0A in resting Ve N/A -enhanced arterial BP responsiveness to hypoxia Gore et al. 200125 6c?E 7c?C Normobaric 15.48% 02 -23 X 9.5h -VO2 during maximal and submaximal exercise N/A N/A N/A N/A N/A -iV02 during submaximal exercise -normoxic exercise Table 1.1 (continued) Summary of Selected Research on intermittent hypoxia and ventilatory control on humans Paper N Intervention Testing HVR HCVR HCVR sb Modified Read Rebreathe V02max Exercise Ventilation Haem. Comments Mahamed & Duffin 200128 5<? 2? Normobaric 20'X70%O2X14d -MRR pre-,post-each exposure -t Ve during resting hypoxia after intervention N/A N/A -left shift in threshold (pre-hypoxia), right-shift post--0Ain sensitivity N/A N/A N/A -increase in resting ventilation during hypoxia and HVD became more apparent Casas et al. 200027 5c? 1? HvDobaric -3-5hX4000-5500Mx17d -haematology N/A N/A N/A N/A -La curve shifted to right -| Ve @ max and La threshold -t PCV, RBC, Hb -climbers Garcia et al. 200010 9c? Hvoobaric (3800m) -2hX 12d -HVR pre- and post--haematology pre-, post--fHVR, peaked on Day 5 then i N/A N/A N/A N/A - 0A in resting Ve in normoxia -| Ve in hypoxia N/A -0A in hb, Hct -fin Ret by Day 5 Garcia et al. 2000" 4c? Hyoobaric (3800m) -2dCHvs. -2h X 2d -HVR pre- and post--fHVR, greater in IH group N/A N/A N/A N/A N/A N/A Rodriguez etal. 199928 8c?C 2?C 6c?E 1?E Kv DO baric -3-5h X 4000-5500m X9d -low intensity exercise in E -V02max, La threshold, haematology -pre-, post-N/A N/A N/A N/A -La curve shifted to R -0Ain V02max -f Ve @ max at sea level -t PCV, RBC, Hb, Ret - 0A b/w C and E groups -climbers Katayama etal. 199829 7c? 6c? HvDobaric (4500m) -exercise:2X 15' @40%VO2max X 6 -both groups 60'X6 -HVR, HCVR, V02max -pre-, post-, 6d post--f in control group -0A in either group N/A N/A -t in training group N/A N/A -HVR used finger Sa02 -t resting Sa02 in control group Table 1.1: Summary of selected research on intermittent hypoxic training and ventilatory control in humans. MRR= Modified Read Rebreathe; '= minutes; h = hours; HVR = hypoxic ventilatory response; HCVR = hypercapnic ventilatory response; HCVRsb = hypercapnic ventilatory response (single-breath); 0A = no change; Haem = haematology; Hb = Haemoglobin concentration; Hct = Haematocrit; RBC = erythrocyte count; Ret = reticulocyte count; Epo = erythropoietin count; E = Experimental; C = Control; SDIH = Short-duration intermittent hypoxia; LDIH = Long-duration intermittent hypoxia; CH = Continuous Hypoxia; Ve = ventilation; V02 = oxygen consumption; La = Lactate; TT = Time Trial Hypobaric vs. Normobaric Intermittent Hypoxia Hypoxia can be hypobaric or normobaric. In hypobaric hypoxia, the fractional concentration of oxygen remains unchanged, while the absolute pressure is decreased. Hypobaric hypoxia requires altitude exposure or a hypobaric chamber. In contrast, normobaric hypoxia involves maintenance of the ambient pressure, but decreasing the inspired oxygen concentration. Most of the research examining the relationship between IH and ventilatory control has been conducted using hypobaric hypoxia, although there have been a few recent studies using normobaric hypoxia 11 23 26 30. Similar changes in HVR have been demonstrated using both protocols. No research has compared the two types of hypoxia in the same study population. Poikilocapnia vs. Isocapnia IH protocols can be either poikilocapnic or isocapnic. The vast majority of studies do not control end-tidal CO2 levels, therefore, during the hypoxic bouts of IH; end-tidal CO2 is decreased, leading to a respiratory alkalosis. Only one study has looked at the effect of isocapnic IH on HVR7 26. Foster et al. showed similar increases in HVR following isocapnic IH to those observed in poikilocapnic studies. Presumably, in isocapnic IH, Jhe respiratory alkalosis would be mitigated, and any changes in respiratory control would be secondary to the hypoxia alone. 8 Hypoxia Duty Cycle Another possible variable that may be important in intermittent hypoxic training is the duty cycle. This term refers to the duration and frequency of each hypoxic exposure. Most published studies in humans have involved a continuous dose of hypoxia each day over several days. This protocol is called continuous intermittent hypoxia or long duration intermittent hypoxia (LDIH). It is not known whether repeated shorter doses of hypoxia each day might provide a more or less profound effect. This type of protocol is termed intermittent-intermittent hypoxia or short-duration intermittent hypoxia (SDIH). Peng and Prabhakar31 examined carotid body hypoxic response in rats following two different protocols of (poikilocapnic) intermittent hypoxia. The SDIH procedure consisted of 15 seconds of normobaric hypoxia (F|02=5%) followed by 5 min of normoxia for 9 episodes per hour, eight hours per day for ten days. The long-duration protocol (LDIH) consisted of 4 hours of hypobaric hypoxia (0.4 atmospheres) per day for ten days. Hypoxic response was significantly enhanced in the SDIH animals but not in the LDIH group. Only one published study has compared the two protocols in humans. Foster et al. 20057, examined chemoreceptor responses in humans exposed to either an SDIH or LDIH protocol but were unable to demonstrate a difference between the two protocols. The study had a few weaknesses: the hypoxic exposures were short (only 30 minutes total daily hypoxia exposure) and occurred as ten exposures over 12 days and subjects were not their own controls. These factors may have 9 increased the variability in response to IH, and thus made it more difficult to demonstrate a difference between SDIH and LDIH. Response to Carbon Dioxide and Intermittent Hypoxia Recently, three separate groups have studied the effects of different IH protocols on the carbon dioxide control of breathing. Interestingly, the results have varied significantly between the three laboratories. Duffin's group26 32 uses a modified rebreathing protocol which incorporates prior hyperventilation to reduce end-tidal carbon dioxide levels (to approximately 20mmHg) before testing. With this technique, one can assess both the CO2 threshold (below which ventilation does not respond to a rise in CO2) and the ventilatory sensitivity to CO2. The experimental paradigm keeps the subject iso-oxic while the carbon dioxide gradually rises as a result of rebreathing. Both hypoxic and hyperoxic conditions are assessed (end-expiratory oxygen pressure of 50 mmHg or 150 mmHg). The hyperoxic trial is intended to reduce the output from the peripheral chemoreceptors33This assertion is controversial, in that Dahan et al.34 examined the response to CO2 in normoxia and hyperoxia. They divided the response into a fast and a slow component, attributing the fast component to the peripheral chemoreceptors. This fast component was reduced but not completely attenuated by hyperoxia. In interpreting the results, therefore, one must consider that there may remain a small residual effect from the peripheral chemoreceptors contributing to the hyperoxic trial. 10 By comparing a hypoxic test where the peripheral chemoreceptors are contributing to respiratory drive to such a hyperoxic test, the investigators can approximate the role of the peripheral chemoreceptors. Using an IH protocol consisting of daily isocapnic twenty-minute exposures to an F|02 of 10%, Mahamed and Duffin examined carbon dioxide response before and after each exposure26. Following the 14-day IH intervention, the subjects demonstrated a leftward shift in the carbon dioxide/ventilation curve. This shift represented a lowering of the carbon dioxide threshold, but no change in carbon dioxide sensitivity. Mateika et al.23 used the same paradigm to examine respiratory response to a shorter IH protocol. Subjects completed eight four-minute bouts at an F|02 of 8% in one session. There was no ensuing change in carbon dioxide threshold, but the sensitivity was increased. These findings are clearly different from Duffin's study, but the IH protocol was also dramatically different. Ainslie et al.4 21 also found an increase in CO2 sensitivity following five nights of hypoxia (4300m). There were some methodological differences between Duffin's protocol (modified Read rebreathing method) and Ainslie's protocol (hyperoxic acute hypercapnic ventilatory response), which make it difficult to make a direct comparison. However, it is interesting that Mateika's study used a shorter IH protocol than Duffin's, and Ainslie's protocol was longer, but they both found an increase in CO2 sensitivity that Duffin did not. Perhaps the fact that Duffin's exposure was the least hypoxic of the three may play a role. Clearly the changes in carbon dioxide sensitivity with IH remain ambiguous. Moreover, no study has looked at both HVR and the modified Read rebreathing method in the same 11 subjects. There may be a possible interaction between changes in CO2 response and changes in HVR. For example, if carbon dioxide threshold is lowered during an iso-oxic test (as a result of IH), ventilation would be increased for a given oxygen concentration. When an HVR test is then performed, this change in C02 threshold could manifest as an increased HVR. It would be therefore be worthwhile to measure CO2 response both before and after ah IH protocol using Duffin's rebreathing method and compare it to the changes that occur in HVR. Respiratory Drive during Exercise Thus far, the ventilatory parameters that have been tracked following IH have been resting values such as HVR and carbon dioxide response. The effect on exercise ventilation is not well understood. Katayama has demonstrated that IH induces an increase in ventilation during hypoxic exercise12 35 (at 40% and 70% of VC«2peak at altitude) but not during sea level (normoxic) exercise13. Conversely, other studies by Casas et al.27 28 and Rodriguez27 28 et al. have demonstrated an increase in ventilation during maximal exercise tests under normoxic conditions following intermittent hypoxia. The IH protocols that the subjects undertook were similar in all three studies except for the fact that Katayama's IH protocol was slightly shorter at seven days (instead of nine or twelve days for the other two studies). Rodriguez' study also had the subjects exercising during their hypoxic exposures, unlike the others which involved passive exposure to hypoxia. Thus, although there is much less work examining 12 ventilatory response to exercise following IH, it appears that ventilation may be increased during hypoxic exercise. During normoxia, there is considerable disagreement. A study examining exercise ventilation under both normoxic and hypoxic conditions is necessary to help resolve this issue. The control of breathing during exercise is multifactorial, while the relationship between the various inputs remains unclear. Arterial blood gases are well-maintained during steady-state below the ventilatory threshold36. Typically, the hyperpnoea of subthreshold exercise follows three phases. At the initial onset of exercise there is a rapid increase in ventilation within one gait cycle. This 'fast component' is likely neurally controlled, either by a central or peripheral stimulus (or a combination of the two)37 38. The second phase of exercise hyperpnoea involves a slower increase in ventilation (the slow component), which is possibly mediated by peripheral chemoreceptors in humans39 40. Eventually, ventilation reaches a steady state, which has been classically described as a summation of the neural (fast) and chemoreceptor (slow) drives to exercise ventilation41. An increase in exercise ventilation resulting from IH would putatively affect the slow component through a similar central neural facilitation mechanism as that proposed for the upregulation of HVR. No studies have examined chemoreceptor response during exercise in humans following an IH protocol. Instead, only resting measures (such as HVR) have been assessed. The process of measuring hypoxic and hypercapnic ventilatory response during exercise is much different than at rest42 43. To measure the hypoxic i 13 response, a subject exercises at steady state and then the inspired gas is switched from air to 100% oxygen for three breaths. The ratio of hyperoxic ventilation to normoxic ventilation is used to indicate the chemoreflex drive to oxygen. A similar mechanism is used to assess hypercapnic response. Instead of oxygen, subjects are given a three-breath stimulus of hypercapnic gas while exercising at steady state. The proposed study would assess both resting and exercising indicators of chemoreflex drive to gain a better understanding of the effect of IH on exercising ventilation. Summary In summary, IH has many effects on the respiratory system that may lead to ergogenic or clinical applications. This study aims to comprehensively assess the resting and exercising control of ventilation while comparing two different types of IH (SDIH and LDIH). The overall aim is to gain a better understanding of the most effective IH protocol and how it affects oxygen and carbon dioxide control of breathing during rest and exercise. 14 RESEARCH QUESTIONS: The proposed study attempts to address the following questions: 1) Is daily measurement of hypoxic ventilatory response a form of intermittent hypoxia? 2) What is the effect of an intermittent hypoxia protocol on resting ventilatory response to hypoxia and hypercapnia? 3) Do two types of intermittent hypoxia protocols (SDIH vs. LDIH) affect the above changes differently? 4) What is the time course of the change in resting ventilatory response to hypoxia as a result of intermittent hypoxia? 5) What is the relationship between changes in hypoxic ventilatory response and changes in both carbon dioxide threshold and sensitivity with intermittent hypoxia? 6) What is the relationship between changes in hypercapnic ventilatory response as measured by the traditional method as compared with the modified Read rebreathing method? 7) Does an intermittent hypoxia protocol affect exercising ventilatory response to hyperoxia and hypercapnia? 8) Does an intermittent hypoxia protocol change submaximal and maximal exercising ventilation under normoxic conditions? 15 9) Does an intermittent hypoxia protocol change submaximal and maximal exercising ventilation under hypoxic conditions? 10) Does an intermittent hypoxia protocol affect exercise capacity in hypoxia? ) 16 HYPOTHESES: 1) Daily measurement of hypoxic ventilatory response would act as an intermittent hypoxic stimulus, thus altering hypoxic ventilatory response. 2) An intermittent hypoxic protocol would result in a co-ordinated increase in oxygen and carbon dioxide chemosensitivity at rest. An intermittent-intermittent hypoxia protocol would lead to larger changes in resting hypoxic ventilatory response and hypercapnic response than the continuous intermittent hypoxia protocol. These changes would occur after fewer days of intermittent hypoxia following the intermittent-intermittent hypoxic protocol, than after the continuous intermittent protocol. 3) An intermittent hypoxic protocol would result in an increase in oxygen and carbon dioxide chemosensitivity during exercise. These augmented responses would lead to increased exercise ventilation under normoxic and hypoxic conditions, improving the arterial saturation and performance during submaximal and maximal hypoxic exercise. i 17 PURPOSE: The purpose of the study was to compare the effects of two different intermittent hypoxia protocols on respiratory chemoresponse and to examine the relationship between carbon dioxide and oxygen sensitivity during rest and exercise. 18 REFERENCES: 1. Rusko HK, Tikkanen HO, Peltonen JE. Altitude and endurance training. J Sports Sci 2004;22(10):928-44; discussion 945. 2. Katayama K, Shima N, Sato Y, Qiu JC, Ishida K, Mori S, et al. Effect of intermittent hypoxia on cardiovascular adaptations and response to progressive hypoxia in humans. High Alt Med Biol 2001 ;2(4):501-8. 3. Greenberg HE, Sica A, Batson D, Scharf SM. Chronic intermittent hypoxia increases sympathetic responsiveness to hypoxia and hypercapnia. J Appl Physiol 1999;86(1):298-305. 4. Kolb JC, Ainslie PN, Ide K, Poulin MJ. Effects of 5 consecutive nocturnal hypoxic exposures on respiratory control and hematogenesis in humans. Adv Exp Med Biol 2004;551:305-10. 5. Zong P, Setty S, Sun W, Martinez R, Tune JD, Ehrenburg IV, et al. Intermittent hypoxic training protects canine myocardium from infarction. Exp Biol Med (Maywood) 2004;229(8):806-12. 6. Golder FJ, Mitchell GS. Spinal synaptic enhancement with acute intermittent hypoxia improves respiratory function after chronic cervical spinal cord injury. J Neurosci 2005;25(11 ):2925-32. 7. Foster GE, McKenzie DC, Milsom WK, Sheel AW. Effects of two protocols of intermittent hypoxia on human ventilatory, cardiovascular and cerebral responses to hypoxia. J Physiol 2005;567(Pt 2):689-99. 8. Townsend NE, Gore CJ, Hahn AG, Aughey RJ, Clark SA, Kinsman TA, et al. Hypoxic ventilatory response is correlated with increased submaximal exercise ventilation after live high, train low. Eur J Appl Physiol 2005;94(1-2):207-15. 9. Katayama K, Sato Y, Shima N, Qiu JC, Ishida K, Mori S, et al. Enhanced chemosensitivity after intermittent hypoxic exposure does not affect exercise ventilation at sea level. Eur J Appl P/7ys/'o/2002;87(2): 187-91. 10. Garcia N, Hopkins SR, Powell FL. Intermittent vs continuous hypoxia: effects on ventilation and erythropoiesis in humans. Wilderness Environ Med 2000; 11 (3): 172-9. 11. Garcia N, Hopkins SR, Powell FL. Effects of intermittent hypoxia on the isocapnic hypoxic ventilatory response and erythropoiesis in humans. Respir Physiol 2000; 123(1 -2):39-49. 19 12. Katayama K, Sato Y, Morotome Y, Shima N, Ishida K, Mori S, et al. Intermittent hypoxia increases ventilation and Sa(02) during hypoxic exercise and hypoxic chemosensitivity. J Appl Physiol 2001;90(4): 1431 -40. 13. Katayama K, Matsuo H, Ishida K, Mori S, Miyamura M. Intermittent hypoxia improves endurance performance and submaximal exercise efficiency. High Alt Med Biol 2003;4(3):291-304. 14. Ling L, Fuller DD, Bach KB, Kinkead R, Olson EB, Jr., Mitchell GS. Chronic intermittent hypoxia elicits serotonin-dependent plasticity in the central neural control of breathing. J Neurosci 2001 ;21(14):5381-8. 15. Mitchell GS, Baker TL, Nanda SA, Fuller DD, Zabka AG, Hodgeman BA, et al. Invited review: Intermittent hypoxia and respiratory plasticity. J Appl Physiol 2001 ;90(6):2466-75. 16. Mitchell GS, Johnson SM. Neuroplasticity in respiratory motor control. J Appl Physiol 2003;94(1 ):358-74. 17. Gallman EA, Millhorn DE. Two long-lasting central respiratory responses following acute hypoxia in glomectomized cats. J Physiol 1988;395:333-47. 18. Foster GE, McKenzie DC, Sheel AW. Effects of enhanced human chemosensitivity on ventilatory responses to exercise. Exp Physiol 2006;91(1):221-8. 19. Katayama K, Sato K, Matsuo H, Hotta N, Sun Z, Ishida K, et al. Changes in ventilatory responses to hypercapnia and hypoxia after intermittent hypoxia in humans. Respir Physiol Neurobiol 2005; 146(1 ):55-65. 20. Katayama K, Sato K, Matsuo H, Ishida K, Iwasaki K, Miyamura M. Effect of intermittent hypoxia on oxygen uptake during submaximal exercise in endurance athletes. Eur J Appl Physiol 2004,92(1-2):75-83. 21. Ainslie PN, Kolb JC, Ide K, Poulin MJ. Effects of five nights of normobaric hypoxia on the ventilatory responses to acute hypoxia and hypercapnia. Respir Physiol Neurobiol 2003; 138(2-3): 193-204. 22. Hendriksen IJ, Meeuwsen T. The effect of intermittent training in hypobaric hypoxia on sea-level exercise: a cross-over study in humans. EurJ Appl Physiol 2003;88(4-5):396-403. 20 23. Mateika JH, Mendello C, Obeid D, Badr MS. Peripheral chemoreflex responsiveness is increased at elevated levels of carbon dioxide following episodic hypoxia in awake humans. J Appl Physiol 2003. 24. Fahlman A, Jackson S, Terblanche J, Fisher JA, Vesely A, Sasano H, et al. A simple breathing circuit to maintain isocapnia during measurements of the hypoxic ventilatory response. Respir Physiol Neurobiol2002;133(3):259-70. 25. Gore CJ, Hahn AG, Aughey RJ, Martin DT, Ashenden MJ, Clark SA, et al. Live high:train low increases muscle buffer capacity and submaximal cycling efficiency. Acta Physiol Scand 2001 ;173(3):275-86. 26. Mahamed S, Duffin J. Repeated hypoxic exposures change respiratory chemoreflex control in humans. J Physiol 2001 ;534(Pt. 2):595-603. 27. Casas M, Casas H, Pages T, Rama R, Ricart A, Ventura JL, et al. Intermittent hypobaric hypoxia induces altitude acclimation and improves the lactate threshold. Aviat Space Environ Med 2000;71 (2): 125-30. 28. Rodriguez FA, Casas H, Casas M, Pages T, Rama R, Ricart A, et al. Intermittent hypobaric hypoxia stimulates erythropoiesis and improves aerobic capacity. Med Sci Sports Exerc 1999;31(2):264-8. 29. Katayama K, Sato Y, Ishida K, Mori S, Miyamura M. The effects of intermittent exposure to hypoxia during endurance exercise training on the ventilatory responses to hypoxia and hypercapnia in humans. Eur J Appl Physiol Occup Physiol 1998;78(3): 189-94. 30. Serebrovskaya TV, Karaban IN, Kolesnikova EE, Mishunina TM, Kuzminskaya LA, Serbrovsky AN, et al. Human hypoxic ventilatory response with blood dopamine content under intermittent hypoxic training. Can J Physiol Pharmacol 1999;77(12):967-73. 31. Peng YJ, Prabhakar NR. Effect of two paradigms of chronic intermittent hypoxia on carotid body sensory activity. J Appl Pfrys/o/2004;96(3): 1236-42; discussion 1196. 32. Duffin J, Mahamed S. Adaptation in the respiratory control system. Can J Physiol Pharmacol 2003;81(8):765-73. 33. Duffin J, Mohan RM, Vasiliou P, Stephenson R, Mahamed S. A model of the chemoreflex control of breathing in humans: model parameters measurement. Respir Physiol 2000; 120(1): 13-26. 21 34. Dahan A, DeGoede J, Berkenbosch A, Olievier IC. The influence of oxygen on the ventilatory response to carbon dioxide in man. J Physiol 1990;428:485-99. 35. Katayama K, Sato Y, Morotome Y, Shima N, Ishida K, Mori S, et al. Cardiovascular response to hypoxia after endurance training at altitude and sea level and after detraining. J Appl Physiol 2000;88(4):1221-7. 36. Ward SA. Control of the exercise hyperpnoea in humans: a modeling perspective. Respir Physiol 2000; 122(2-3): 149-66. 37. Koehle M, Duffin J. The effect of exercise duration on the fast component of exercise hyperpnoea at work rates below the first ventilatory threshold. Eur J Appl Physiol Occup Physiol 1996;74(6):548-52. 38. Bell HJ. Respiratory control at exercise onset: An integrated systems perspective. Respir Physiol Neurobiol 2006;152(1):1-15. 39. Whipp BJ. Peripheral chemoreceptor control of exercise hyperpnea in humans. Med Sci Sports Exerc 1994;26(3):337-47. 40. Prabhakar NR, Peng YJ. Peripheral chemoreceptors in health and disease. J Appl Physiol 2004;96(1 ):359-66. 41. Miyamoto Y. Neural and humoral factors affecting ventilatory response during exercise. Jpn J Physiol 1989;39(2): 199-214. 42. Cooper T. Peripheral Chemoresponsiveness and Exercise Induced Arterial Hypoxemia in Highly Trained Endurance Athletes. [Master's Thesis]. University of British Columbia, 1993. 43. Fukuoka Y, Endo M, Oishi Y, Ikegami H. Chemoreflex drive and the dynamics of ventilation and gas exchange during exercise at hypoxia. Am J Respir Crit Care Med 2003;168(9):1115-22. 22 CHAPTER 2: REPEATED MEASUREMENTS OF HYPOXIC VENTILA TORY RESPONSE AS AN INTERMITTENT HYPOXIC STIMULUS? A version of this chapter has been published as: Koehle MS, Foster GE, McKenzie DC and Sheel AW (2005) Repeated measurement of hypoxic ventilatory response as an intermittent hypoxic stimulus Resp Physiol Neurobiol 145(1): 33-39. 23 RESEARCH QUESTION: 1) Is daily measurement of hypoxic ventilatory response a form of intermittent hypoxia? METHODS: The study was approved by the University of British Columbia's Clinical Research Ethics Board. Nine male subjects were recruited. Subjects were screened by a physician for a history of respiratory disease, cardiovascular disease or smoking. They were not taking any medication during the study period. Mean subject age was 26.7 ±6.2 years (mean ±SD). Subjects visited the lab on a total of six occasions over a ten-day period. The visits took place on days 1, 6, 7, 8, 9 and 10. Subjects were asked to refrain from caffeine and ethanol intake and moderate exercise in the six hours prior to the laboratory tests. On the first visit, subjects were informed of the details and protocol for the study and informed consent was obtained. Resting spirometry was then performed according to the standards of the American Thoracic Society1 to exclude occult respiratory disease by using a portable spirometer (Spirolab II, Medical International Research, Rome, Italy). Following spirometry, subjects underwent a resting isocapnic HVR test. This initial HVR test acted as the baseline test to which the repeated tests would later be compared. On subsequent test days, subjects came to the lab and relaxed quietly for a minimum often minutes before HVR testing. 24 The HVR testing protocol was based on an earlier method2"6. Subjects breathed through a respiratory mask (Hans-Rudolph 8980, Kansas City, MO, USA) attached to a one-way non-rebreathing valve (Hans-Rudolph 2700, Kansas City, MO, USA). Inspiratory flow was measured using a heated pneumotach (Hans-Rudolph HR800, Kansas City, MO, USA). Arterial 02 saturation was measured using pulse oximetry at the finger (Model 503, CSI Criticare Systems Inc., Waukesha, WI, USA). End-tidal Pco2 was sensed at the mouth using a C02 sensor (Model CD-3A, Applied Electrochemistry, Pittsburgh, PA, USA). Inspired O2 concentration was sampled upstream of the non-rebreathing mask using an 02 sensor (Model S-3-A/I, Applied Electrochemistry, Pittsburgh, PA, USA). A 13.5-litre mixing chamber was located upstream of the pneumotach. The manual addition of varying flows of 100% N2 to the mixing chamber allowed control of F|02. Ventilatory and gas values (flow, tidal volume, frequency, Sa02, Pco2, F|02) were displayed in real time during testing (PowerLab, ADI Instruments, Colorado Springs, CO, USA). Inspired volume was calculated using the integrated flow signal and the frequency of breathing. Data were sampled at 400 Hz. The subjects rested in a supine position and breathed room air for five minutes in a darkened room while listening to ambient music to reduce external stimuli that could affect respiration. The test period started when 100% N2 was introduced into the inspired gas mixture. The flow of N2 was started at 2 litres per minute and increased at a rate of one litre per minute every 30 seconds. This protocol gradually lowered inspired O2 concentration to approximately 5% over a period of approximately five minutes. To maintain isocapnia, CO2 was added to 25 the inspired mixture using a manually controlled valve. The investigator monitored the displayed end-tidal PCO2 value and adjusted the flow of CO2 added to the inspired gas mixture, just proximal to the non-rebreathing mask. The test was terminated once the arterial saturation fell below 80%. Data and Statistical Analysis Ventilatory data were acquired on a breath-by-breath basis. The last minute of rest was used to calculate the resting ventilatory values. Ventilation was then plotted against saturation. Using the trendline function, a best-fit slope was plotted by computer (Microsoft Excel, Redmond, WA, U.S.A.). The absolute value of the slope was taken as the hypoxic ventilatory response. Using statistical software (STATISTICS 6.1, Stat Soft Inc., Tulsa OK, USA), resting tidal volume, frequency, minute ventilation and HVR were analysed using repeated measures analysis of variance (ANOVA) procedures, with time as the independent variable. Linear regression was used to find a trend in hypoxic ventilatory response over the five sequential test days. A p-value of 0.05 was used to determine statistical significance. RESULTS: Anthropometric data and spirometry are presented in Table 2.1. Mean resting ventilatory parameters (minute ventilation, frequency, tidal volume and end-tidal PCO2) are included in Table 2.2. No significant differences were noted 26 between the sample days for any of the resting parameters, (p-values were 0.75, 0.81, 0.94 and 0.83 for minute ventilation, frequency, tidal volume and end-tidal PCO2, respectively). The mean intra-individual coefficient of variation for resting end-tidal Pco2 was 4%. Mean ±SD % Predicted Age (years) 26.8 ±6.1 Height (centimetres) 181.0 ±5.8 Mass (kilograms) 76.3 ±6.1 Forced Vital Capacity (FVC) (litres) Forced Expired Volume (FEV1.0) (litres) 5.60 ±0.97 4.64 ±0.92 103 ±13 100 ±16 FEV/FVC (%) 82.6 ±4.0 99 ±6 Table 2.1: Anthropometric and spirometry data for all subjects. Data are expressed as means ±SD. 27 Day Frequency (breaths-min"1) Tidal Volume (litres) Minute Ventilation (litresmin"1) End-tidal Pco2 (mmHg) Hypoxic Ventilatory Response (litres-min" 1%Sa02"1) 1 12.9 ±4.3 0.92 ±0.31 10.56 ±2.42 41.6 ±2.1 0.70 ±0.58 (0.12-2.04) 6 13.2 ±3.1 0.90 ±0.20 11.04 ±2.00 43.1 ±2.1 0.70 ±0.40 (0.36- 1.58) 7 13.6 ±3.9 0.92 ±0.22 11.60 ±2.46 42.2 ±2.1 0.70 ±0.59 (0.23-2.23) 8 14.2 ±3.2 0.85 ±0.24 11.44 ±1.19 41.9 ±4.0 0.68 ±0.38 (0.36-1.56) 9 14.2 ±3.7 0.91 ±0.23 12.36 ±3.53 42.4 ±6.0 0.80 ±0.72 (0.40 - 2.63) 10 13.6 ±3.1 0.86 ±0.17 11.43 ±3.52 42.7 ±2.1 0.66 ±0.43 (0.33-1.71) Mean 13.6 ±3.6 0.89 ±0.23 11.40 ±2.52 42.3 ±3.3 0.71 ±0.51 Table 2.2: Resting respiratory parameters and hypoxic ventilatory response (HVR). Values are expressed in means ±SD. HVR range is shown in parentheses. Figure 2.1 demonstrates a sample tracing from a single representative HVR test. Two minutes of resting data are demonstrated. The beginning of the test occurs once the F|Oz begins to decrease from its baseline value. As the haemoglobin saturation decreases following the F|02, the ventilation gradually increases. In general, the increased minute ventilation was mediated through an 28 increase in both frequency and tidal volume, but the contribution of tidal volume seemed to be the larger of the two components. 1 15 10 1.6 r 30 60 90 120 150 180 210 -240 270 300 330 360 390 420 Time: Figure 2.1: Sample data from one HVR test on one subject. 29 The HVR plot derived from the sample tracing in Figure 2.1 is shown in Figure 2.2. The x-axis (saturation) is plotted from right-to-left (with 100% saturation on the left) by convention to display a positive slope. Figure 2.3 demonstrates the individual HVR values for each of the subjects on each of the test days. Inter- and intra-individual variation was present in these values, but there was no obvious trend in HVR. Mean HVR values (with standard deviations) are presented in Table 2.2. There were no significant differences in HVR between any of the test days (p=0.86). Regression failed to show any trend in HVR over the five sequential days (p=0.97). The calculated mean coefficient of variation for HVR for each subject was 27%. 30 20 4 2 100 95 90 85 80 75 Sao2 (%) Figure 2.2: HVR plot using the data from Figure 2.1. The x-axis (saturation) is plotted right-to-left by convention. 31 DISCUSSION: To determine the optimal duration and frequency of intermittent hypoxia required to achieve the greatest change in HVR, daily measurements of HVR are required. Similarly, to measure the changes in HVR during acclimatisation to continuous hypoxia, daily measurements are desirable. To date, human studies that have involved repeated measurements of HVR during IH or acclimatisation (over days) 7 8 have not included a control group (i.e. subjects not exposed to episodic or intermittent hypoxia, but still had their HVR measured daily). Therefore, the independent effect of repeated measurements of HVR is unknown. This study is the first to examine whether repeated measurement of 32 HVR does in fact change HVR. The results failed to show a trend over 5 days of repeated HVR measurements, or a difference between the five measurements and a control measurement taken 5 days prior. The short exposure to hypoxia as part of HVR measurement is, therefore, likely not a co-intervention when measured repeatedly (24 hours apart) in physiological studies of acclimatisation and intermittent hypoxic training in humans. This study also provides information about the repeatability of HVR measurements. HVR is a notoriously variable parameter9"12. Sahn et al.9 compared intra-individual variability in HVR, and demonstrated that intra-day variability was much less than between-day variability. The coefficient of variation (CV) was 19% for intra-day measurements and between-day variability was 1.2-15 times greater than within-day variability. Zhang and Robbins10 examined between-day variability in HVR (measurements were at least one week apart) and found a coefficient of variation of 26%. Other studies examining multiple HVR measurements in the same subject have shown much more variability,911 with CVs reported as high as 76%. To our knowledge, there have been no published studies that have examined repeatedly measured HVR over several consecutive days (in the absence of other stimuli). Previous reports of between-day HVR variability have been at least a week apart. The mean coefficient of variation for the present experiment was 27% (±13), comparable to the 26% reported by Zhang and Robbins10. Both studies measured HVR on six occasions in each subject. Unique to our study was the measurement of HVR over five consecutive days. Since the CV was comparable 33 between the two studies, it makes us more confident that repeated measurement of HVR does not lead to an independent augmentation of HVR. Furthermore, the methodology used in the present study represents a repeatable paradigm for assessing HVR which demonstrates a coefficient of variation that is comparable to previously published data 911. Methodological Considerations Arterial oxygen saturation was measured by pulse oximetry instead of by arterial catheterisation. Pulse oximetry has potential disadvantages that may influence the results. This method was chosen because it is non-invasive, portable and has a negligible complication rate as compared to arterial catheterisation. Pulse oximetry can be influenced by poor peripheral perfusion, as can occur during exercise, cold and Raynaud's phenomenon. In the present study, all these factors were excluded. Additionally, pulse oximetry fails to compensate for changes in pH and temperature which both affect the haemoglobin-oxygen dissociation curve. As subjects were at rest during the present study, there were no changes in temperature during the course of an HVR measurement. Changes in pH were minimised by strict control of end-tidal PC02 during testing. The hypoxic exposures in this study involved isocapnic hypoxia. Although many intermittent hypoxic protocols utilise poikilocapnic hypoxia, there have also been studies documenting ventilatory changes following isocapnic intermittent 34 hypoxia . As HVR is most often measured using an isocapnic hypoxic stimulus, we chose isocapnia for this study. CONCLUSIONS Repeated measurement of HVR does not lead to a change in HVR itself. Therefore, repeated HVR measurement does not act as a significant co-intervention in short-term acclimatisation and intermittent hypoxia studies. The HVR method used in the present study demonstrates comparable reproducibility to previously published data. 35 REFERENCES 1. Standardization of Spirometry, 1994 Update. American Thoracic Society. Am J Respir Crit Care Med 1995;152(3):1107-36. 2. Hopkins SR, McKenzie DC. Hypoxic ventilatory response and arterial desaturation during heavy work. J Appl Physiol 1989;67(3):1119-24. 3. Weil JV, Byrne-Quinn E, Sodal IE, Friesen WO, Underhill B, Filley GF, et al. Hypoxic ventilatory drive in normal man. J Clin Invest 1970;49(6):1061-72. 4. Cherniack NS, Dempsey J, Fend V, Fitzgerald RS, Lourenco RV, Rebuck AS, et al. Workshop on assessment of respiratory control in humans. I.Methods of measurement of ventilatory responses to hypoxia and hypercapnia. Am Rev Respir Dis 1977;115(1):177-81. 5. Bartsch P, Swenson ER, Paul A, Julg B, Hohenhaus E. Hypoxic ventilatory response, ventilation, gas exchange, and fluid balance in acute mountain sickness. High Alt Med Biol 2002;3(4):361 -76. 6. Guenette JA, Diep TT, Koehle MS, Foster GE, Richards JC, Sheel AW. Acute hypoxic ventilatory response and exercise-induced arterial hypoxemia in men and women. Respir Physiol Neurobiol 2004; 143(1):37-48. 7. Garcia N, Hopkins SR, Powell FL. Intermittent vs continuous hypoxia: effects on ventilation and erythropoiesis in humans. Wilderness Environ Med 2000;11(3):172-9. 8. Sato M, Severinghaus JW, Bickler P. Time course of augmentation and depression of hypoxic ventilatory responses at altitude. J Appl Physiol 1994;77(1):313-6. 9. Sahn SA, Zwillich CW, Dick N, McCullough RE, Lakshminarayan S, Weil JV. Variability of ventilatory responses to hypoxia and hypercapnia. J Appl Physiol 1977;43(6): 1019-25. 10. Zhang S, Robbins PA. Methodological and physiological variability within the ventilatory response to hypoxia in humans. J Appl Physiol 2000;88(5): 1924-32. 11. Fahlman A, Jackson S, Terblanche J, Fisher JA, Vesely A, Sasano H, et al. A simple breathing circuit to maintain isocapnia during measurements of the hypoxic ventilatory response. Respir Physiol Neurobiol 2002; 133(3):259-70. 12. Beidleman BA, Rock PB, Muza SR, Fulco CS, Forte VA, Jr., Cymerman A. Exercise VE and physical performance at altitude are not affected by menstrual cycle phase. J Appl Physiol 1999;86(5): 1519-26. 36 13. Morris KF, Gozal D. Persistent respiratory changes following intermittent hypoxic stimulation in cats and human beings. Respiratory Physiology & Neurobiology 2004; 140(1): 1 -8. 14. Bavis RW, Mitchell GS. Plasticity in Respiratory Motor Control: Selected Contribution: Intermittent hypoxia induces phrenic long-term facilitation in carotid-denervated rats. JApp/P/7ys/o/2003;94(1):399-409. 15. Mahamed S, Duffin J. Repeated hypoxic exposures change respiratory chemoreflex control in humans. J Physiol 2001 ;534(Pt. 2):595-603. 16. Baker TL, Mitchell GS. Episodic but not continuous hypoxia elicits long-term facilitation of phrenic motor output in rats. J Physiol 2000;529 Pt 1:215-9. 37 CHAPTER 3: INTERMITTENT HYPOXIA AND ITS EFFECT ON RESTING MEASURES OF CHEMORESPONSE 38 RESEARCH QUESTIONS: 1) What is the effect of an intermittent hypoxia protocol on resting ventilatory response to hypoxia and hypercapnia? 2) Do two types of intermittent hypoxia protocols (short duration intermittent hypoxia and a long duration intermittent hypoxia) affect the above changes differently? 3) What is the time course of the change in resting ventilatory response to hypoxia as a result of intermittent hypoxia? 4) What is the relationship between changes in hypoxic ventilatory response and changes in both carbon dioxide threshold and sensitivity with intermittent hypoxia? 5) What is the relationship between changes in hypercapnic ventilatory response as measured by the traditional method as compared with the modified Read rebreathing method? METHODS: The study was approved by the University of British Columbia Clinical Research Ethics Board. A non-blinded randomised crossover study design was used. Subjects were evaluated before, during and following two intermittent hypoxia protocols: a short duration intermittent hypoxia (SDIH) and a long duration intermittent hypoxia (LDIH) protocol. The study paradigm is depicted in Figure 3.1. Ten male subjects were recruited from the University population and the local mountaineering and endurance sports community. The subjects were 39 healthy, recreationally active and had not travelled to altitude in the preceding 6 months. All subjects were asked to come to the laboratory a total of 19 times, totalling approximately 40 hours each. These visits included one orientation visit, seven visits for short-duration intermittent hypoxic exposure, seven visits for long-duration intermittent hypoxic exposure and four follow-up measurement sessions. 40 Visit # 1 -Informed consent -Familiarisation with equipment -Pulmonary Function testing -HVR -HCVR -Modified Rebreathing Test -Graded exercise test in normoxia Figure 3.1: Study Paradigm LDIH Arm Visit # 2 -HVR -HCVR -Modified Rebreathing Test -IH 60 min Visit # 3 -HVR -IH 60 min Visit #4 -HVR -IH 60 min Visit # 5 -HVR -IH 60 min Visit # 6 -HVR -IH 60 min Visit #7 -HVR -IH 60 min TWO WEEKS BETWEEN RANDOMISED ARMS OF THE STUDY Visit #8 -HVR -IH 60 min Visit # 9 -HVR -HCVR -Modified Rebreathi ng Test Visit #10 45 min -HVR -HCVR -Modified Rebreathing Test SDIH Arm Visit #11 -HVR -HCVR -Modified Rebreathing Test -IH 110 min Visit #12 -HVR -IH 110 min Visit # 13 -HVR -IH 110 min Visit # 14 -IH 1.10 min Visit # 15 -HVR -IH 110 min Visit # 16 -HVR -IH 110 min Visit #17 -HVR -IH 110 min Visit* 18 -HVR -HCVR -Modified Rebreathi ng Test Visit # 19 -HVR -HCVR -Modified Rebreathing Test On the initial visit, informed consent was obtained, followed by baseline spirometry and familiarisation with the equipment. Subjects filled out a physical activity screening questionnaire1 (PAR-Q, CSEP, www.csep.ca, Canada). At least one HVR test and two modified rebreathing tests were performed to acquaint the subjects with the testing protocol and equipment. Subjects also performed a normoxic maximal oxygen uptake test at this time on an electronically braked cycle ergometer (Lode, Groningen, Netherlands). A ramp protocol was used with wattage starting at 0 W and increasing by 0.5 W/s until volitional fatigue. Inspiratory flow was measured using a heated pneumotach (Hans-Rudolph, Kansas City, MO, USA). Minute ventilation was calculated using the integrated flow signal and the frequency of breathing. Arterial oxygen saturation was measured using pulse oximetry at the finger. Expired gases were collected in a mixing chamber and analysed using a carbon dioxide and an oxygen sensor (Applied Electrochemistry, Pittsburgh, PA, USA). Ventilatory and gas values were displayed in real time during testing (PowerLab, ADI Instruments, Colorado Springs, CO, USA). Respiratory values were averaged every 15s. The highest 4 consecutive values were averaged to determine maximal values. Peak power at the end of exercise was recorded. Hypoxic Ventilatory Response Resting ventilatory tests were performed in a quiet environment with distractions minimised. The HVR testing protocol followed the protocol used in 42 previous studies in the laboratory2 3 and is based on an earlier method used in other facilities4. It is described in detail in Chapter 2 of this dissertation. Briefly, subjects breathed through a respiratory mask attached to a one-way non-rebreathing valve (Hans-Rudolph, Kansas City, MO, USA). Inspiratory flow was measured using a heated pneumotach (Hans-Rudolph, Kansas City, MO, USA). Minute ventilation was calculated using the integrated flow signal and the frequency of breathing. Arterial oxygen saturation was measured using pulse oximetry at the finger. End-tidal carbon dioxide and inspired oxygen concentrations were measured on a breath-by-breath basis using a carbon dioxide and an oxygen sensor (Applied Electrochemistry, Pittsburgh, PA, USA). Ventilatory and gas values were displayed in real time during testing (PowerLab, ADI Instruments, Colorado Springs, CO, USA). During the entire HVR test, subjects listened to quiet, ambient music through headphones. The subjects rested in a supine position while breathing room air for five minutes. The resting end-tidal carbon dioxide was determined from the last minute of this rest period. The test started when 100% nitrogen was introduced into the inspired gas mixture. The flow of nitrogen increased at rate of one litre per minute every 30 seconds. This protocol gradually lowered inspired oxygen concentration from 21% to approximately 5% over a period of five minutes. To maintain isocapnia, carbon dioxide was added to the inspired mixture using a manually controlled valve. The test ended once the arterial saturation reached 80%. Ventilation was then plotted against saturation, with the absolute value of the magnitude of the slope representing the HVR. 43 Breath-by-breath ventilation was then plotted against saturation. A best-fit slope was plotted by computer using the built-in trendline function in Microsoft Excel (Redmond, WA, U.S.A). Hypercapnic Ventilatory Response Test The hypercapnic ventilatory response (HCVR) testing protocol was based on the protocol of Katayama et al.5 This test involved no prior hyperventilation. Following the HVR testing, subjects rested in a seated position prior to their HCVR test for approximately 5 minutes. Wearing nose clips, subjects breathed room air ad libitum through a three-way rebreathing valve (Hans-Rudolph, Kansas City, MO, USA) that was connected to a rebreathing bag. This bag was filled with 7% N2, balance O2. Inspiratory flow was measured using a heated pneumotach (Hans-Rudolph, Kansas City, MO, USA). Inspired volume was calculated using the integrated flow signal and the frequency of breathing. Arterial oxygen saturation was measured using pulse oximetry at the finger. End-tidal carbon dioxide and inspired oxygen concentrations were assessed on a breath-by-breath basis using a carbon dioxide and an oxygen sensor (Applied Electrochemistry, Pittsburgh, PA, USA). Ventilatory and gas values were displayed in real time during testing (LabVIEW 7.0, National Instruments, Austin, TX, USA). After reaching a steady-state resting ventilation, subjects exhaled completely before they were switched over to the rebreathing bag. They took 44 three large breaths to equilibrate the gas in their lungs with that in the bag. Subjects were then asked to breathe ad libitum. The test was terminated once PETC02 reached 60mmHg, minute ventilation reached 100 litres per minute or in the instance of subject discomfort. HCVR sensitivity was determined as the slope of minute ventilation (L»min1) plotted against end-tidal CO2 (mmHg). Modified Rebreathing Test The modified rebreathing testing protocol was based on the protocol of Read6 and modified by Duffin7 8. At the start of each test, subjects hyperventilated for five minutes to reduce their end-tidal CO2 partial pressure to between 19 and 25 mmHg. They were coached during this rebreathing period to maintain this desired end-tidal CO2 level. Using a rebreathing valve (Hans-Rudolph, Kansas City, MO, USA), subjects were then switched to breathe into a bag that was filled with a mixture of 42mmHg C02 and either 50mmHg or 150mmHg of oxygen (for the hypoxic and hyperoxic tests, respectively). The rebreathing bag was maintained iso-oxic using a computer-controlled valve9 (LabVIEW 7.0, National Instruments, Austin, TX, USA) while end-tidal C02 was allowed to progressively rise. The test was performed twice, with 02 pressures maintained at either 50 mmHg (hypoxic condition) or 150 mmHg (hyperoxic condition). The test was terminated once PCO2 reached 60mmHg, minute ventilation reached 100 litres per minute or in the instance of subject discomfort. Using specifically designed software10, the data from each test was used to calculate CO2 threshold and sensitivity. The model parameters are described in 45 detail in Duffin et al. 2000.11 Briefly, the software fits a straight line to the C02/time relationship and derives a predicted C02 for each breath based on this model. Ventilation is then plotted against this predicted CO2, and the model then fits three line segments to the rebreathing data. The first segment follows the exponential decline to basal ventilation following the cessation of hyperventilation. The second segment begins at the first breakpoint, the slope of which is reported as the sensitivity. If there is a second breakpoint, a third segment is plotted beyond this point. Breakpoints and other parameters are adjusted in an iterative manner to optimise fit via minimisation of the sum of squares (Levenberg-Marquardt algorithm)12. Using the technique, one will often find that above the threshold, the CCVventilation relationship can appear to consist of two segments (Figure 3.2), the first one is more gradual and is mainly mediated through increases in tidal volume, whereas the second slope seems to be more frequency-mediated11. The sensitivity calculated using this software is for the first (lower) slope. As the second segment was not uniformly present, it was not assessed in the present analysis. 46 80 -, 0 -I , . , , —\ , . 1-—-, 1 . , , 1 . . , . 1 30 35 40 45 50 Predicted end-tidal C02 (mmHg) Figure 3.2: Sample data from an individual modified rebreathing test demonstrating the CO2 threshold (dashed vertical line) and the two subsequent slopes in the COyventilation relationship. For the purpose of this study, the first slope was assessed to determine CO2 sensitivity. Intermittent Hypoxic Training The final task for the subjects on their second visit day was their intermittent hypoxic training. Normobaric hypoxic gas (12% O2, balance N2) was provided by mask. For the LDIH protocol, subjects breathed the hypoxic gas for 60 minutes daily for seven days. This protocol was chosen because it was the normobaric equivalent of that used in Katayama's study that showed the increases in exercise ventilation during hypoxia13. For the SDIH protocol, the subjects spent 115 minutes breathing from the mask each day. They alternated 47 through 12 cycles of 5 minutes of hypoxia (simulated 4400 metres) followed by 5 minutes of normoxia. Subjects were then required to return to the lab for 6 additional IH sessions. These were identical to the session on the first day. Each IH exposure was preceded by measurement of isocapnic HVR. Follow-up The first day following the final IH protocol the subjects returned for Post-IH testing. This testing was exactly the same as the pre-testing. It consisted of an isocapnic HVR test, an HCVR test and two modified rebreathing tests (hypoxic and hyperoxic). One week following the completion of the first round of IH, subjects returned to the laboratory for 7-days Post-IH testing. This session included HVR testing, an HCVR test and two modified rebreathing tests. Subjects were given at least two weeks washout between each arm of the study (range 14-94 days). Two weeks was chosen because previous studies had shown that HVR remained somewhat elevated at one week after intermittent hypoxia13, but not two weeks post-IH14. Pre- and Post- testing was the same in both arms of the study; the only difference was the nature of the intermittent hypoxic training (SDIH or LDIH). The entire visit paradigm is demonstrated in Figure 3.1. 48 Statistics Each of these resting tests (HVR, HCVR, Modified Rebreathing) were compared at three time points (Pre-, Post- and 7 days Post-IH) using ANOVA with repeated measures over time and IH protocol has an independent factor. Where the null hypothesis was rejected, Tukey's HSD was calculated to determine the significant differences. Data are presented as means (±SD). Linear correlations were also performed between HVR and CO2 threshold (Pre-, Post- and Delta). Statistical analysis was performed using computer software (SPSS Inc., Chicago, IL, U.S.A.); an alpha of 0.05 was used to determine statistical significance. Using previously reported data, using a very similar intermittent hypoxic protocol,13 with a mean post-protocol HVR of 0.71 ±0.15 L-min'^/oSacV1, to detect a 25% difference in HVR (SDIH vs. LDIH, a= 0.05 and B= 0.80) a sample size of 9.586 was required. RESULTS: All ten subjects completed all parts of the study. Anthropometric data, respiratory and exercise data are presented in Table 3.1. 49 Parameter Mean Standard Deviation Age (years) 26.0 6.7 Height (cm) 177.2 8.1 Mass (kg) 72.8 13.9 Forced Vital Capacity - (FVC) 5.84 0.70 % Predicted FVC 112.9 9.8 Forced Expired Volume (1-second) (FEV1.0) 5.01 0.61 % Predicted (FEV1.0) 114.2 9.1 FEV/FVC (%) 85.9 3.8 % Predicted FEV/FVC (%) 101.5 4.0 Maximal Oxygen Consumption (Absolute) (L«min1) 4.23 0.82 Maximal Oxygen Consumption (Relative) (mL»kg"1»min1) 58.2 3.9 Maximal Power (Watts) 335 67 Table 3.1: Anthropometric, Respiratory and Exercise Baseline Data. FVC = forced vital capacity, FEVi.o = forced expired volume in one litre. Hypoxic Ventilatory Response HVR results are presented in Figure 3.3. Mean baseline HVR for both protocols was 0.47 (±0.23) L»min"1»%Sao2"1. After the 7-day IH protocols, HVR was increased by 93 (±120%) and 65% (±74%) (for LDIH and SDIH, respectively). This difference was significant (p<0.01 and p<0.05, for LDIH and SDIH respectively). The difference between the two protocols was not significant. One week post-IH, HVR remained non-significantly elevated with 50 both protocols. Individual data are presented in Figures 3.4 and 3.5. When protocol order was examined (to assess for potential learning effect) there was no difference in the change in HVR between the protocol that the subjects performed chronologically first and that which they performed second (Figure 3.6). * - significantly different from Pre- (p< 0.05) ** - sianif icantlv different from Pre- (DO.OD 6 7 8 9 Time (Protocol Day) • LDIH D SDIH 10 11 12 13 14 Figure 3.3: Mean (±SD) Hypoxic Ventilatory Response (HVR) vs. Time. The measurement on Day 1 occurs prior to the first hypoxic exposure. The measurement on Day 8 occurred the first day following intermittent hypoxia. 51 Time (Protocol Day) Figure 3.4: Individual Hypoxic Ventilatory Response (HVR) vs. Time during the LDIH Protocol. Thick black line denotes mean response. The measurement on Day 1 occurs prior to the first hypoxic exposure. The measurement on Day 8 occurred the first day following intermittent hypoxia. 52 012345678 Time (Protocol Day) Figure 3.5: Individual Hypoxic Ventilatory Response (HVR) vs. Time during the SDIH Protocol. Thick black line denotes mean response. The measurement on Day 1 occurs prior to the first hypoxic exposure. The measurement on Day 8 occurred the first day following intermittent hypoxia. 1.4 i 1.2 Time (Protocol Day) Figure 3.6: Mean (±SD) Hypoxic Ventilatory Response (HVR) vs. Time by Protocol Order. The measurement on Day 1 occurs prior to the first hypoxic 53 exposure. The measurement on Day 8 occurred the first day following intermittent hypoxia. Both protocols caused non-significant increases in resting ventilation with the increase from LDIH (12.7 ±19.9%, p=0.074), more pronounced than that following SDIH (8.4 ±15.9%, p=0.13). There was no significant difference between the two protocols. Modified Rebreathing In both the hyperoxic and hypoxic modified rebreathing tests, the CO2 sensitivity was unchanged by either protocol of IH. In hypoxia, the CO2 threshold was significantly reduced following both protocols. LDIH reduced the threshold by 1.60 (±0.98) mmHg, whereas following SDIH it was reduced by 1.98 (±2.60) mmHg. Under hyperoxic conditions, LDIH reduced the CO2 threshold by 2.06 (±2.33) mmHg, and SDIH caused a reduction of 2.53 (±1.36) mmHg. There were no significant differences between the two protocols. At 7 days following the IH, these threshold values were still lower than baseline (but not significantly so). These results are displayed in Figures 3.7 and 3.8. 54 Pre- Post- 7 Days Post-* - significantly different from Pre- (p< 0.01) Figure 3.7: Mean (±SD) Carbon dioxide Threshold in Hyperoxia vs. Time (Po2 150 mmHg). • LDIH • SDIH Post-* - significantly different from Pre- (p< 0.05) ** - significantly different from Pre- (p<0.01) 7 Days Post-Figure 3.8: Mean (±SD) Carbon dioxide Threshold in Hypoxia vs. Time (Po2 = 50 mmHg). 55 Hypercapnic Ventilatory Response The Hypercapnic Ventilatory Response (HCVR) was significantly increased by IH by 42.9 (±63.4)% (p<0.01). This value remained elevated by 38.1 (±70.9)% at 7 days following IH (p<0.01). When analysed by protocol, HCVR was increased significantly by the LDIH protocol by 56.1 (±71.6)% (p<0.01) and remained elevated by 54.4 (±94.0)% at 7 days post (p<0.01). The changes following the SDIH protocol were smaller at 29.7 (±54.6)% and 21.9 (±34.4)%, at 1 and 7 days post IH, respectively. The increases following SDIH were not significant, nor were the differences between the two protocols. These data are presented in Figure 3.9. 56 7 • LDIH • SDIH Pre- Post- 7 Days Post^ * - significantly different from Pre- (p< 0.01) Figure 3.9: Mean (+SD) Hypercapnic Ventilatory Response (HCVR) vs. Time. Associations between Chemosensitivity Measures When HVR, measured prior to the first performed protocol was compared with the values from the Duffin technique, no significant correlations were found for CO2 threshold (r= -0.499 and -0.625, for hypoxic and hyperoxic conditions, respectively) or sensitivity (r= 0.137 and 0.233). Secondly, when the change in HVR was compared with the change in C02 threshold by the Duffin technique, no significant correlations were found (r= -0.058 and 0.091). When HCVR was compared with CO2 sensitivity by the Duffin method (in hyperoxia), there was no significant relationship (p=0.532). 57 DISCUSSION: Ten male subjects, acting as their own controls, underwent two consecutive different IH protocols in a random order.. Resting measurements of HVR, HCVR, CO2 threshold and CO2 sensitivity were performed prior to, immediately following, and one week following the IH. HVR measurements were also performed daily during the protocol. Intermittent hypoxia increased HVR by approximately 50% while decreasing the threshold to CO2 in hypoxia and hyperoxia. HCVR was also increased following IH. All the volunteers were healthy and recreationally active. The mean relative Vo2max was 58.2 mL«kg~1»min~\ with a standard deviation of only 3.9. Thus in terms of fitness, these subjects represent a relatively homogeneous group with above average fitness for a recreationally active cohort. Hypoxic Ventilatory Response The observed increases in HVR are consistent with previous work that showed comparable changes in HVR with similar IH protocols1516. This is the first study to measure the HVR daily over 7 consecutive days of IH. From these measurements it appears that majority of the augmentation of resting HVR following these protocols occurs in the first 4 days. The HVRs from the fourth and the eighth days were not statistically different. This finding may indicate that shorter IH protocols may be adequate if the goal of IH is augmentation of HVR. 58 Hypercapnic Ventilatory Response The relationship between IH and augmentation of HCVR has been much less consistent. In earlier work, Katayama et al.13 showed no change in HCVR after 7 days of poikilocapnic IH (60 minutes per day), but more recently they demonstrated an increase after a 14 day protocol (3 hours per day)17. Ainslie et al.18 were also able to demonstrate an increase in the slope of HCVR following 5 nights of 8-9 hours of poikilocapnic IH. Using isocapnic IH (with only 30 minutes per day of hypoxia), Foster15 showed no difference in HCVR. It appears that the studies that incorporate longer durations of poikilocapnic hypoxia tend to affect HCVR whereas those that maintain isocapnia or employ shorter bouts of hypoxia do not augment HCVR. Longer and poikilocapnic exposures may cause a more profound, prolonged hypocapnic stimulus to increase the sensitivity to CO2. Modified Rebreathing Using the modified Read rebreathing tests, we were able to examine both the CO2 threshold and the CO2 sensitivity. The test was performed under both hypoxic and hyperoxic conditions. In theory, the hyperoxic trial attenuates the contribution from the peripheral chemoreceptors to preferentially target the central chemoreceptors as discussed previously. We found that CO2 threshold was reduced following both IH protocols in hypoxia and hyperoxia. In the only other study to examine IH and CO2 threshold19, subjects were exposed to 14 consecutive daily exposures to 20 minutes of isocapnic hypoxia. Mahamed and Duffin found a decrease in threshold only under the hypoxic condition and not the hyperoxic 59 hyperoxic condition, attributing this alteration to the effects of intermittent hypoxia on the peripheral chemoreceptor in the absence of any change in CO2. The current study differs in that the exposures were longer and were poikilocapnic; no study had previously looked at the effects of poikilocapnic IH on CO2 response by the Duffin technique. Mahamed et al.12 showed that the repeated hypoxic hypercapnic exposures of obstructive sleep apnoea caused an overnight increase in sensitivity to C02 (in the hyperoxic test) but no change in threshold. They found no changes in the hypoxic rebreathe test. In summary, it appears that intermittent hypoxia has variable effects on the CO2 sensitivity and threshold in hypoxia and hyperoxia that depend on the level of CO2 (poikilo-, iso- or hypercapnic) and the duration and severity of the hypoxia. A study that compares the C02 responses to intermittent exposures to a given dose of hypoxia but under poikilocapnic, isocapnic and hypercapnic conditions is required to clarify the role of C02 level on the effect of IH on C02 response. In the current study, sensitivity to hypercapnia was increased following IH in the HCVR test, but not the hyperoxic modified rebreathing test. Furthermore, there was no correlation between HCVR and CO2 sensitivity by the Duffin technique. This study is the first to compare HCVR and the Duffin technique following the same intervention, but is not the first time that discordant results have been obtained with the two measurements. Fuse et al20 and Mahamed et al.12 both looked at overnight changes in response to hypoxic hypercapnia in patients with obstructive sleep apnoea using the HCVR and the Duffin method, respectively. Fuse found no change in HCVR overnight, while in a later study, 60 Mahamed found an increase in CO2 sensitivity. Mahamed attributed this discrepancy to the fact that the Duffin method measures CO2 sensitivity over a different range of end-tidal CO2 than the HCVR method. Because the hyperventilation (used in Duffin's technique) reduces end-tidal C02 to a subthreshold level, the slope of the CO2 sensitivity was measured from a lower point (by about 4 to 8 mmHg) than in the HCVR. This lower starting point becomes even lower following IH. Such a difference becomes important if the C02/ventilation relationship is not truly linear. In the tests where more than one sensitivity slope was evident, the lower slope (starting at the threshold) is assessed by the analysis software. As HCVR slope assessment occurs at higher CO2 levels, these two assessments may not overlap as much as one would initially expect. Thus, the HCVR may be assessing response at higher partial pressures of CO2 than the Duffin technique, leading to the different outcomes that were observed. This is the not the first study to compare two measurements of C02 sensitivity and find differing results. Pandit et al.21 compared CO2 sensitivity by the steady-state and the Read rebreathing methods (without prior hyperventilation), and found that the sensitivity response was steeper in the rebreathing method. They also found differing effects on cerebral blood flow sensitivity to carbon dioxide between the two methods. Variations in cerebral blood flow may play a role in the differing responses to CO2 observed in the present study. Hyperventilation reduces cerebral blood flow through its effect on 61 cerebral vasodilatation. This reduced cerebral blood flow could alter the tissue Pco2, and thus the response of the central chemoreceptors. The 5 minutes of hyperventilation in the Duffin technique may cause other inputs to the control of breathing which are not present in HCVR measurement. For example, in some individuals, the 5 minutes of voluntary hyperventilation can induce a short-term potentiation of ventilation22. Furthermore, there may be behavioural inputs to ventilation following hyperventilation that may affect the result. Datta et al.23 showed that ventilation following a period of hyperventilation to induce hypocapnia is affected by wakefulness. Subjects that were asleep showed a longer, more consistent apnoea following hyperventilation than while awake. The authors concluded that other behavioural drives affect ventilation during this period. Thus, cerebral blood flow, STP or behavioural drives to breathing may act as further modulators of ventilation, diluting the effect of an alteration in CO2 sensitivity from IH. As the HCVR technique does not involve hyperventilation, it would not be subject to these other influences. In summary, although both the HCVR and the modified rebreathing method each assess a form of CO2 sensitivity, the results are not equivalent. This discordance between HCVR and the modified Read rebreathing method may also relate to the CO2 levels at which the sensitivity is assessed (higher with HCVR), or different inputs to ventilation brought about by the 5 minutes of prior hyperventilation. One way to evaluate the role of the prior hyperventilation would be to use a eucapnic voluntary hyperpnoea protocol, whereby the subject increases their ventilation but by inspiring a mixed gas 62 containing C02, they do not become hypocapnic. The behavioural and STP components of the hyperventilation would be maintained but CO2 sensitivity would be measured in the range of the HCVR. The advantage of the HCVR is that it measures central chemoresponsiveness without the confounding effects of the 5-minute prior rebreathe. The modified rebreathing technique is able to determine the C02 threshold under both hypoxic and hyperoxic conditions. Thus if threshold or peripheral chemoresponsiveness is the most important outcome, the modified rebreathing technique is most appropriate, while if central chemoreceptor response to CO2 is the variable of interest, HCVR would be more appropriate. SDIH vs. LDIH There was no difference between SDIH and LDIH for any of the measured variables. As with the other studies of IH in humans, there is a large amount of inter- and intra-individual variation in the chemosensitivity measures2. This factor makes it more difficult to notice subtle differences between protocols. We therefore may not be able to rule out a small difference in efficacy between SDIH and LDIH, but a large (and arguably physiologically significant) difference between them is unlikely. Foster et al.15 had similar findings when comparing SDIH and LDIH. In their study, the subjects did not act as their own controls, increasing the potential for variation. Furthermore, the IH protocol was 5 days on, two days off, 5 days on. This led to a somewhat irregular protocol causing a more uneven profile of HVR augmentation. The doses of IH in the current study 63 were also approximately double (in daily duration) that of Foster et al. Finally, in the study of animals that demonstrated- the increased efficacy of SDIH over LDIH, Peng and Prabhakar24 used poikilocapnic hypoxia, unlike Foster's group (who used isocapnic hypoxia). The current study used poikilocapnic hypoxia, which better replicates the animal work. These four factors should make the current study design more sensitive to a difference between SDIH and LDIH. Consequently, the absence of a difference in the current study should make one more confident of a lack of benefit of SDIH over LDIH in affecting resting chemosensitivity in humans. SDIH and LDIH differ in the number of hypoxic on-transients and off-transients. The duration of SDIH (5-minute bouts) chosen in the present study was chosen to mitigate the effects of hypoxic ventilatory decline (HVD) on the stimulus to ventilation during the intermittent hypoxia. Theoretically, each bout was short enough to end before HVD could become a significant factor. Conversely, the LDIH protocol would expose the subjects to HVD each day. The finding that SDIH was not more efficacious than LDIH lends support to the concept that in humans, the number of transitions from hypoxia to normoxia may not be as important as the total exposure time to hypoxia. Furthermore, avoidance of any HVD effect may not be instrumental in stimulating augmentation of chemoresponse following IH. 64 Limitations Oxygen saturation was estimated by pulse oximetry at the finger for all tests. The concordance between oxygen saturation at rest by pulse oximetry and by arterial blood gas measures is reasonable at saturations above 85%.25 At saturations below this value, the accuracy of these devices deteriorates. The HVR protocol used in the current study required monitoring the subjects to an oxygen saturation of 80%. Presumably, the accuracy during the final portion of the HVR would be reduced, increasing the error. Unfortunately, because breath-by-breath monitoring of oxygen saturation is required, pulse oximetry is necessary (as direct measures could not provide the breath-by-breath values in real time). This factor has the potential to increase the variability in the HVR measurements and reduces the power of the study. Another potential criticism is that the washout period may have been inadequate. Recent work from Katayama17, suggests that if two IH protocols are done consecutively, the HVR might increase sooner in the second than in the first protocol (indicating a form of metaplasticity). To assess whether the length of the washout period was adequate, we compared the daily HVRs from the first and second protocols (chronologically) and found no significant differences. In Katayama's study, the daily IH exposures were 3 times as long as in the present study, which may mean that a more prolonged daily dose is required to cause this potentiation of HVR response to IH. A sample size of 10 was chosen to detect a 25% difference between SDIH and LDIH post-protocol. Thus, a difference between the two protocols of less 65 than 25% would not have been detected. Thus there could have been a small difference between the two protocols that was undetected by the current study. CONCLUSIONS: Following two different 7-day IH protocols administered to subjects in a crossover fashion, there were increases in HVR and HCVR, along with a left shift in the C02 threshold in both hypoxia and hyperoxia. The majority of the augmentation in HVR occurred by the fourth day of the IH. No differences occurred between the SDIH and LDIH protocols in terms of respiratory response at rest. The poikilocapnic IH protocol appeared to cause more potentiation of the central chemoreceptors (as measured by HCVR and hyperoxic rebreathing methods) than in previous studies using shorter doses of isocapnic IH. 66 REFERENCES: 1. Thomas S, Reading J, Shephard RJ. Revision of the Physical Activity Readiness Questionnaire (PAR-Q). Can J Sport Sci 1992;17(4):338-45. 2. Koehle MS, Foster GE, McKenzie DC, Sheel AW. Repeated measurement of hypoxic ventilatory response as an intermittent hypoxic stimulus. Respir Physiol Neurobiol 2005; 145(1 ):33-9. 3. Guenette JA, Diep TT, Koehle MS, Foster GE, Richards JC, Sheel AW. Acute hypoxic ventilatory response and exercise-induced arterial hypoxemia in men and women. Respir Physiol Neurobiol 2004;143(1):37-48. 4. Cherniack NS, Dempsey J, Fend V, Fitzgerald RS, Lourenco RV, Rebuck AS, et al. Workshop on assessment of respiratory control in humans. I.Methods of measurement of ventilatory responses to hypoxia and hypercapnia. Am Rev Respir Dis 1977;115(1): 177-81. 5. Katayama K, Sato Y, Ishida K, Mori S, Miyamura M. The effects of intermittent exposure to hypoxia during endurance exercise training on the ventilatory responses to hypoxia and hypercapnia in humans. Eur J Appl Physiol Occup Physiol 1998;78(3): 189-94. 6. Read DJ. A clinical method for assessing the ventilatory response to carbon dioxide. Australas Ann Med 1967;16(1):20-32. 7. Mohan R, Duffin J. The effect of hypoxia on the ventilatory response to carbon dioxide in man. Respir Physiol 1997;108(2):101-15. 8. Duffin J, Mahamed S. Adaptation in the respiratory control system. Can J Physiol Pharmacol 2003;81(8):765-73. 9. Boston Flow Rebreathing 2003 [Windows program]. 4 version. Toronto, 2004. 10. Analyse Rebreathing Full.vi [Windows program]. v20 Toronto: Duffin J., 2004. 11. Duffin J, Mohan RM, Vasiliou P, Stephenson R, Mahamed S. A model of the chemoreflex control of breathing in humans: model parameters measurement. Respir Physiol 2000; 120(1): 13-26. 12. Mahamed S, Hanly PJ, Gabor J, Beecroft J, Duffin J. Overnight changes of chemoreflex control in obstructive sleep apnoea patients. Respir Physiol Neurobiol 2005;146(2-3):279-90. 1.3. Katayama K, Sato Y, Morotome Y, Shima N, Ishida K, Mori S, et al. Intermittent hypoxia increases ventilation and Sa(02) during hypoxic exercise and hypoxic chemosensitivity. J Appl Physiol 2001 ;90(4):1431-40. 14. Katayama K, Sato Y, Morotome Y, Shima N, Ishida K, Mori S, et al. Ventilatory chemosensitive adaptations to intermittent hypoxic exposure with endurance training and detraining. J Appl Physiol 1999;86(6): 1805-11. 67 15. Foster GE, McKenzie DC, Milsom WK, Sheel AW. Effects of two protocols of intermittent hypoxia on human ventilatory, cardiovascular and cerebral responses to hypoxia. J Physiol 2005;567(Pt 2):689-99. 16. Katayama K, Sato Y, Shima N, Qiu JC, Ishida K, Mori S, et al. Enhanced chemosensitivity after intermittent hypoxic exposure does not affect exercise ventilation at sea level. Eur J Appl Physiol 2002;87(2): 187-91. 17. Katayama K, Fujita H, Sato K, Ishida K, Iwasaki K, Miyamura M. Effect of a repeated series of intermittent hypoxic exposures on ventilatory response in humans. High Alt Med Biol 2005;6(1):50-9. 18. Ainslie PN, Kolb JC, Ide K, Poulin MJ. Effects of five nights of normobaric hypoxia on the ventilatory responses to acute hypoxia and hypercapnia. Respir Physiol Neurobiol 2003; 138(2-3): 193-204. 19. Mahamed S, Duffin J. Repeated hypoxic exposures change respiratory chemoreflex control in humans. J Physiol 2001 ;534(Pt. 2):595-603. 20. Fuse K, Satoh M, Yokota T, Ohdaira T, Muramatsu Y, Suzuki E, et al. Regulation of ventilation before and after sleep in patients with obstructive sleep apnoea. Respirology 1999;4(2): 125-30. 21. Pandit JJ, Mohan RM, Paterson ND, Poulin MJ. Cerebral blood flow sensitivity to C02 measured with steady-state and Read's rebreathing methods. Respir Physiol Neurobiol 2003;137(1):1-10. 22. Chatha D, Duffin J. The pattern of breathing following a 10-breath voluntary hyperventilation during hyperoxic rebreathing. Can J Appl Physiol 1997;22(3):256-67. 23. Datta AK, Shea SA, Horner RL, Guz A. The influence of induced hypocapnia and sleep on the endogenous respiratory rhythm in humans. J Physiol 1991;440:17-33. 24. Peng YJ, Prabhakar NR. Effect of two paradigms of chronic intermittent hypoxia on carotid body sensory activity. J Appl Physiol 2004,96(3): 1236-42. 25. Kolb JC, Farran P, Norris SR, Smith D, Mester J. Validation of pulse oximetry during progressive normobaric hypoxia utilizing a portable chamber. Can J Appl Physiol 2004;29(1):3-15. 68 CHAPTER 4: INTERMITTENT HYPOXIA AND ITS EFFECT ON EXERCISE CHEMOSENSITIVITY 69 RESEARCH QUESTIONS: 1) Does an intermittent hypoxia protocol affect exercise performance in hypoxia? 2) Does an intermittent hypoxia protocol affect exercising ventilatory response to hyperoxia and hypercapnia? 3) Does an intermittent hypoxia protocol change submaximal and maximal exercising ventilation under normoxic conditions? 4) Does an intermittent hypoxia protocol change submaximal and maximal exercising ventilation under hypoxic conditions? 5) Do two types of intermittent hypoxia protocols (short duration intermittent hypoxia and a long duration intermittent hypoxia) affect the above changes differently? METHODS This study was approved by the University of British Columbia Research Ethics Board. It was conducted in conjunction with the experiment described in Chapter 3 of this dissertation. The same crossover study design, intermittent hypoxia protocol and subjects were used for both studies. Using exercise testing, subjects were evaluated prior to and following two intermittent hypoxia protocols: a short duration intermittent hypoxia (SDIH) and a long duration intermittent hypoxia (LDIH) protocol. Ten male subjects were recruited from the University population and the local mountaineering and endurance sports community. All subjects were asked to come to the laboratory a total of 17 times. These visits 70 included one orientation visit, seven visits for short-duration intermittent hypoxia and seven visits for long-duration intermittent hypoxia and two follow-up measurement sessions. On the initial visit, informed consent was obtained, followed by baseline spirometry and familiarisation with the equipment. Subjects filled out a physical activity screening questionnaire12 (PAR-Q, CSEP, Canada). All exercise testing was performed on an electronically braked cycle ergometer (Lode, Groningen, Netherlands). Subjects also performed a normoxic maximal oxygen uptake test at this time. The initial normoxic graded exercise test was used to determine the subsequent submaximal work rates (the protocol for this test is previously described in Chapter 3 of this thesis). For the submaximal exercise test, work rates corresponding to 35% and 60% of the normoxic maximal work rate were used. (These values are expected to correspond to approximately 40% and 70% of maximal work rate breathing 15% O2, based on previous research conducted in our laboratory.)3 Exercise Test The exercise test consisted of three components: a normoxic submaximal test, a hypoxic submaximal test and a hypoxic graded exercise test. The normoxic and hypoxic components were separated by ten minutes of rest. Two work rates were used for each submaximal test. Low intensity corresponded to 35% of the normoxic maximal work rate, while moderate intensity corresponded to 60% of the normoxic maximal work rate. The exercise test protocol is depicted 71 in Figure 4.1. 100 n -10 0 10 20 30 40 50 60 70 Time (minutes) Figure 4.1: Exercise Testing Protocol - shaded area indicates hypoxic condition The protocol used to assess the chemosensitivity during exercise was based on method previously used in this laboratory4 and others5. After a self-selected warm-up, subjects commenced with the normoxic submaximal exercise test. They pedalled at 35% of normoxic maximal work rate for 10 minutes. During the final seven minutes of this exercise bout they received two hyperoxic and two hypercapnic stimuli. Each of these stimuli was separated by approximately two minutes (ventilation had always returned to the pre-stimulus level by approximately one minute post-stimulus). Steady-state ventilation was determined from the mean ventilation for each 30-second period prior to each chemoreceptor stimulus. The hyperoxic test consisted of abruptly switching the inspired gas from room air to 100% oxygen for 3 breaths before reverting to room air. To estimate the hyperoxic chemosensitivity during exercise, the mean 72 minute ventilation during the 30 seconds prior to the stimulus was compared to the nadir of the three-second moving average of the breath-by-breath minute ventilation post-stimulus. For the hypercapnic test, the inspired mixture was switched to 10% carbon dioxide (21% oxygen, balance nitrogen) for one single breath. To estimate the hypercapnic chemosensitivity during exercise the mean minute ventilation during the 30 seconds prior to the stimulus was compared to the peak of the three-second moving average of the breath-by-breath minute ventilation post-stimulus. The order of the hyperoxic and hypercapnic stimuli was randomised. The subjects were blinded to this order. Following ten minutes and two bouts of both hyperoxia and hypercapnia the low-intensity submaximal test was complete. Without stopping exercise, work rate was then increased to 60% of normoxic maximal work rate. Subjects then performed the same protocol a second time at this new work rate. At the conclusion of the normoxic submaximal work rate subjects rested for approximately ten minutes before starting the hypoxic exercise test. The hypoxic mixture consisted of 15% oxygen (balance nitrogen) provided by mask at atmospheric pressure. This oxygen concentration was chosen because it was the lowest concentration at which subjects can consistently finish a maximal exercise test without reaching an arterial oxygen saturation below 70% by pulse oximetry3. Before starting to exercise in hypoxia, subjects rested for approximately 2 to 3 minutes while breathing the hypoxic mixture. The hypoxic submaximal exercise test followed the same protocol as the normoxic submaximal test. For the hypercapnic chemosensitivity test, subjects breathed 73 one breath of 10% carbon dioxide with 15% oxygen and balance nitrogen in order to maintain the F|02 constant while only altering the F|C02. The hyperoxic stimulus was unchanged from normoxic exercise (at 100% 02 for three breaths). Immediately following the hypoxic submaximal test, subjects transitioned to a graded maximal oxygen uptake test (still in hypoxia). At this point, the gas sample line was transferred from the mouthpiece (for breath-by-breath analysis) to a mixing chamber to allow for determination of oxygen consumption. Intermittent Hypoxia Protocol The final task for the subjects on their second visit day was their intermittent hypoxic exposure. Normobaric hypoxic gas (12% 02, balance N2) was provided by mask. For the LDIH protocol, subjects breathed the hypoxic gas for 60 minutes daily for seven days. For the SDIH protocol, the subjects spent 115 minutes breathing from the mask each day. They were exposed to 12 cycles of 5 minutes of hypoxia (simulated 4400 metres) followed by 5 minutes of normoxia. The order of the SDIH and LDIH arms of the study was randomised but not blinded. Subjects were required to return to the lab for 6 subsequent IH sessions. Follow-up The first day following the final IH protocol the subjects returned for follow-up exercise testing. This testing protocol was the same as in the pre-test. 74 Following a washout period of at least 2 weeks, subjects entered the second arm of the study, undergoing the IH protocol that they had not already completed. Data Analysis All exercise variables were assessed at two time points, on the first day of IH (Pre-IH) and on the first day following the completion of the IH protocol (Post-IH). Variables were compared using analysis of variance with repeated measures over time. Significance was set at p<0.05. Data are presented as means (±SD). RESULTS All subjects attended all laboratory sessions and performed all tests (to the best of their ability). No subjects left the study for any reason. Submaximal Exercise Test Minute ventilation was unchanged during both normoxic and hypoxic submaximal exercise following IH. These data are presented in Table 4.1. Condition Intensity LDIH SDIH Pre-IH Post-IH Pre-IH Post-IH Normoxia Low 40.4 ±8.5 41.2 ±7.6 41.4 ±8.5 41.4 ±7.8 Moderate 68.6 ±12.1 70.1 ±11.0 72.1 ±15.6 71.6 ±11.4 Hypoxia Low 46.3 ±9.9 47.3 ±8.7 48.8 ±9.4 48.9 ±10.3 Moderate 91.6 ±19.4 89.4 ±14.1 93.2 ±17.4 92.2 ±17.3 Table 4.1: Exercise Minute Ventilation Pre- and Post-Intermittent Hypoxia (IH). Values are expressed in L«min"1 (±SD). 75 The hyperoxic trials caused a transient nadir in minute ventilation following 3 breaths of 100% oxygen. The decrease in minute ventilation following hyperoxic challenge was significantly higher in moderate intensity exercise than in low intensity exercise (p<0.01). The hyperoxic test also caused larger changes in ventilation in hypoxia than in normoxia (p<0.01). The effect of the hyperoxic tests on minute ventilation increased in magnitude following IH (p< 0.05) from 14.4 (±9.5) to 15.3 (±9.9). L»min"1. There was no difference between the two protocols. Data for each condition and protocol are presented in Table 4.2. Condition Intensity LDIH SDIH Pre-IH Post-IH Pre-IH Post-IH Normoxia Low 7.0 ±4.1 7.1 ±4.4 6.2 ±4.1 6.2 ±4.6 Moderate 10.7 ±5.9 11.6 ±6.5 11.0 ±5.1 12.5 ±4.5 Hypoxia Low 13.7 ±6.5 15.2 ±5.9 16.2 ±6.5 17.3 ±6.9 Moderate 24.5 ±7.6 27.1 ±10.7 26.1 ±10.7 27.3 ±7.3 Mean 14.0 ±8.9 14.7 ±10.1 14.9 ±10.1 15.6 ±9.6 Table 4.2: Mean decreases in Minute Ventilation following 3 breaths of hyperoxia (Pre- and Post-IH). Values are expressed in % (±SD). Results of the hypercapnic challenge were similar to those of the hyperoxic challenge. In contrast to the hyperoxic results, there were no differences in the decrease in the ventilation following the hypercapnic challenge as a result of the intermittent hypoxia (p<0.01 for both). Hypercapnia had more 76 of an effect in moderate than low intensity exercise and a larger effect in hypoxia than in normoxia (p<0.01 for both). Data for each condition and protocol are presented in Table 4.3. Condition Intensity LDIH SDIH Pre-IH Post-IH Pre-IH Post-IH Normoxia Low 8.8 ±4.1 12.5 ±7.1 8.6 ±4.1 13.6 ±5.4 Moderate 10.7 ±5.9 11.6 ±6.5 13.6 ±5.4 13.9 ±6.8 Hypoxia Low 12.6 ±6.1 11.5 ±6.7 12.8 ±6.8 13.3 ±7.8 Moderate 17.3 ±5.9 15.3 ±6.9 13.6 ±6.6 17.0 ±7.9 Mean 12.8 ±6.5 12.2 ±6.7 12.1 ±6.1 13.7 ±7.2 Table 4.3: Mean increases in Minute Ventilation following 1 breath of hypercapnia (Pre- and Post-IH). Values are expressed in % (±SD). Pulse oximetry results were unchanged during submaximal normoxic exercise following an IH protocol. In hypoxia, there was a significant increase (p<0.01) in mean arterial oxygen saturation following IH (both protocols combined). This increase was highly significant following the SDIH protocol, but not significant following the LDIH protocol. (Figure 4.2) The difference between the two protocols was not statistically significant. To test the relationship between resting HVR and exercising ventilation in hypoxia, a correlation was performed between HVR and the change in minute ventilation between normoxic submaximal exercise and hypoxic submaximal exercise. No relationship was evident (r=0.024 and p=0.947). 77 LDIH SDIH Intermittent Hypoxia Protocol Combined Figure 4.2: Mean (±SD) Oxygen Saturation (%) during Submaximal Hypoxic Exercise vs. Intermittent Hypoxia Protocol. **-denotes statistically significant (p <0.01) from Pre- value. Hypoxic Graded Exercise Test Of the 10 subjects, 8 were able to complete the exercise protocol each time, including some portion of the hypoxic graded exercise test at the conclusion of the hypoxic submaximal exercise test. The two subjects who were unable to complete the entire exercise protocol stopped due to volitional exhaustion. For tests where the subjects were unable to complete the entire submaximal portion of the test, their highest submaximal wattage was taken as the peak wattage for that particular exercise test. Following IH, there were no changes in peak wattage, peak ventilation or peak oxygen consumption. These 78 data are presented in Figure 4.3. There were no significant differences between the two protocols. LDIH SDIH Combined Intermittent Hypoxia Protocol Figure 4.3: Mean (±SD) Peak Wattage vs. Intermittent Hypoxia Protocol. Combined refers to the collective data for both SDIH and LDIH protocols. Of the 8 subjects who completed the entire exercise test, there were only complete oxygen consumption data on 5 of those subjects (due to difficulties switching the gas sampling from breath-by-breath to mixed gases mid-test). Following IH, peak oxygen consumption in hypoxia was 3.7 (±9.7)% higher, but this difference was not significant (p=0.12). This increase was similar with both protocols (displayed in Figure 4.4). Peak ventilation was unchanged following the LDIH protocol, but increased by 10.9 (±14.7) % after the SDIH protocol. Peak ventilation data are presented in Figure 4.5. 79 LDIH SDIH Combined Intermittent Hypoxia Protocol Figure 4.4: Mean (±SD) Peak Oxygen Consumption (L»min1) vs. Intermittent Hypoxia Protocol. Combined refers to the collective data for both SDIH and LDIH protocols. LDIH SDIH Combined Intermittent Hypoxia Protocol Figure 4.5: Mean (±SD) Peak Exercise Ventilation (L«min~1) vs. Intermittent Hypoxia Protocol. Combined refers to the collective data for both SDIH and LDIH protocols. 80 DISCUSSION: Ten male subjects completed two different IH protocols in a random order, acting as their own controls. Submaximal exercise measurements of ventilation, saturation and response to hypercapnic and hyperoxic stimuli were assessed in normoxia and hyperoxia. A modified graded exercise test in hypoxia was also performed. All measurements were performed prior to and following IH. Following IH, small increases in arterial oxygen saturation during submaximal exercise were observed. IH did not affect minute ventilation, peak power or oxygen consumption during peak exercise. The responses to hyperoxic or hypercapnic stimuli during submaximal exercise were not significantly altered by IH, although the increase in response to the hyperoxic stimulus approached significance. Minute Ventilation During Normoxic Exercise Submaximal minute ventilation was unchanged following IH in both the normoxic and hypoxic conditions. Under normoxic conditions, many previous studies have shown no increase in submaximal exercise ventilation 6"9. Using a similar IH protocol, the current study corroborates these findings. Conversely, two studies employing longer IH protocols1011 found an increase in submaximal minute ventilation during normoxic exercise following IH. The protocols involved 23 and 19 nights of 8-10 hours of hypoxic exposures that were slightly hypobaric (F|02= 15.48% and 16.3% with Patm ~710 mmHg). Townsend11 found that the augmentation in ventilation was already apparent after the first four nights of IH 81 exposure. In these "live-high, train-low" protocols, subjects were sleeping in hypoxic enclosures (for 8-10 hours) as opposed to breathing from a mask while awake for only one hour each day. Two possible explanations exist for the difference between these two types of studies. One possibility would be that the longer durations of hypoxia might provide a more substantial stimulus to exercise ventilation that was only apparent in the overnight studies. Secondly, sleeping during hypoxia may have a different effect on ventilation than remaining awake during hypoxia. While alert, the subjects would have more ventilatory stimuli and would have a higher arterial oxygen saturation for a given level of hypoxia. Periodic breathing occurs in hypoxia12, but would be more common in sleeping subjects than in awake subjects. An increase in periodic breathing could also lead to more profound hypoxaemia (and a stronger IH stimulus) in the sleeping subjects than if they were awake. Thus, these "live-high, train-low" studies might provide a greater hypoxic stimulus than those of Katayama and the current study leading comparatively larger increases in HVR11. As exercise ventilation is controlled by a larger number of factors than resting ventilation, the effect of augmented HVR on exercising ventilation could be obscured by these other factors. Consequently, a larger increase in HVR (by longer exposures during sleep) may be required to cause measurable changes in submaximal exercise ventilation. More research is needed to compare awake to sleeping IH and to assess the effects of longer daily doses of hypoxia. 82 Minute Ventilation During Hypoxic Exercise In the one previous study to look at submaximal exercise under hypoxic conditions, Katayama showed a significant increase in submaximal exercise ventilation under hypobaric hypoxic conditions13. In that study, subjects exercised at 432 mmHg (approximately equivalent to an F|02 of 11.9%). In contrast, our subjects exercised in normobaria at an F|02 of 15%. The exercise type (cycle ergometer) and duration in both studies was 10 minutes each at low and moderate intensities, respectively. Even the exercise intensities were comparable; in Katayama's study, low and moderate intensity corresponded to 40 and 70% of hypoxic Vo2peak, while in the current study, low and moderate intensity corresponded to 35 and 60% of normoxic Vo2peak. The IH protocols (60 minutes/day for 7 days) were very close, except that in the Katayama study the exposures were hypobaric. There are two potential explanations for the discrepancy between the two studies: the effects of hypobaria, and the timing of the hypoxic exposures with the onset of exercise. The differential effects of normobaric hypoxia and hypobaric hypoxia are unclear. Two studies in humans have shown that resting ventilation in normobaric hypoxia is higher than in an equivalent level of hypobaric hypoxia14 15. If an equivalent augmentation of exercising ventilation occurred in normobaric hypoxia, over hypobaric hypoxia, it could possible explain the discrepancy. That is to say, higher baseline ventilation occurring during normobaric hypoxic exercise could mask an effect of IH on exercise ventilation. 83 In a hypobaric chamber, to bring subjects to the desired simulated altitude requires approximately 30 minutes; subjects therefore would have a short period of progressive hypoxia prior to the onset of exercise. In the current (normobaric) study, subjects breathed hypoxic gas at rest for only 2-3 minutes prior to the start of exercise. Thus, in the two studies, subjects' submaximal exercise ventilations were tested at different time points during an acute exposure to hypoxia. Hypoxic Ventilatory Decline (HVD) refers to the decrease in the acute ventilatory response to hypoxia that occurs within 5 to 30 minutes of exposure to hypoxia16. Presumably, in the normobaric hypoxic study, subjects would have started exercising prior to the onset of HVD, while in the hypobaric study; the measurements would not have commenced until after this decline had already occurred. HVD has been described only during rest, but this attenuation of ventilatory response could also occur during exercise. If IH acted to mitigate the effects of HVD on exercising ventilation, its effect would only be evident if the exercise took place following the onset of HVD (as in Katayama's study) and not irt the present study. In summary, IH does not appear to augment exercising ventilation in normobaric hypoxia as it does during hypobaric hypoxia, possibly because the effects of hypobaria on ventilation or the presence of an HVD phenomenon in the hypobaric study. 84 Hyperoxic Test The 3-breath doses of 100% oxygen caused transient decreases in ventilation that were more pronounced during the hypoxic condition and the moderate exercise intensity. IH significantly enhanced these changes. The purpose of this hyperoxic challenge test was to remove the hypoxic drive to ventilation from the peripheral chemoreceptors. The difference between the nadir in post-hyperoxic stimulus ventilation and the mean pre-stimulus ventilation was ascribed to the hypoxic drive from the peripheral chemoreceptors during exercise. These findings could indicate that augmentation of hypoxic ventilatory response at rest leads to a concomitant increase in the magnitude of the hypoxic drive during exercise. The findings do not improve our understanding of the location of the modulation in hypoxic respiratory control. Evidence indicates that either the carotid bodies themselves, or central facilitation of chemoreceptor afferents are modulated through a metaplastic process.17 18. Either mechanism (or a combination of both) could potentially augment the chemoreceptor afferent input to exercise ventilation. One would expect that if resting hypoxic ventilatory response played a significant role in the control of the hyperpnoea of exercise, that those with a higher resting HVR would have a larger augmentation in ventilation when transitioned from normoxic to hypoxic exercise. This situation did not occur, in the present study. Furthermore, the lack of effect on submaximal minute ventilation would indicate that although this hypoxic drive to breathe may have been increased, that it's effect on total ventilation during exercise is modest. 85 Work by Sheel et al. compared resting HVR to maximal ventilation during a graded exercise test. No relationship was found, indicating that resting HVR is not a major determinant of peak exercise ventilation. There are two potential reasons for the unchanged minute ventilation in the face of augmented hypoxic drive to exercise. The augmented hypoxic drive could alter the pattern of ventilation without changing the minute ventilation. For example, when HVR is increased by chronic hypoxic exposure to altitude, there is an augmentation of tidal volume20 preferentially over breathing frequency. In the current study, although minute ventilation was unchanged, there could have been an increased in tidal volume that was not apparent when minute ventilation data was compared. This change would have the effect of enhancing alveolar ventilation without increasing minute ventilation. Alternatively, the increased hypoxic drive to ventilation during exercise is of too small a magnitude to increase minute ventilation. The size of the ventilatory transients following hyperoxic challenge was only increased by about 6%. Considering the magnitude of other neural and humoral inputs to exercise hyperpnoea, this altered chemoresponse could be just to small to lead to a perceptible effect. Further analysis of the pattern of breathing during exercise is warranted to understand the interaction between enhanced hypoxic drive and minute ventilation. 86 Hypercapnic Test To test the effect of IH on C02-medlated control of breathing during exercise, subjects were given single breath doses of 10% CO2 at several points during their submaximal exercise tests. These hypercapnic breaths caused a transient increase in ventilation that was unaffected by IH. At rest, IH caused a left shift in the CO2 threshold that would increase the ventilation at a given end-tidal C02 pressure. This study is the first to modulate response to C02 during rest, and then to test its effect during exercise. There were no differences in the effect of the hypercapnic tests following IH, suggesting that the augmentation of resting CO2 response following IH did not lead to significant changes in C02 control of exercise ventilation. The changes in ventilation following the hyperoxic and hypercapnic tests were larger in moderate exercise than in low intensity exercise. The load on the ergometer was independent of cadence. Because subjects were encouraged to maintain their cadence constant throughout the exercise test, workload would have limited the effect of mechanically sensitive peripheral limb afferents on exercise hyperpnoea. The modulation of exercise hyperpnoea would therefore have been mediated by a variety of central, humoral, behavioural and factors. At the moderate intensity, the magnitude of the hypoxic drive was increased, indicating that its effect on exercise hyperpnoea was undiminished as compared to low intensity exercise. These changes in ventilation were also more pronounced in hypoxia than in normoxia. Presumably in hypoxic exercise, the 87 contribution of the hypoxic drive to exercise minute ventilation would be more important. Removing this drive with hyperoxia would then cause a larger drop in ventilation. Oximetry during Submaximal Exercise Arterial oxygen saturation was obtained using pulse oximetry, which has limitations during exercise22, and must be interpreted cautiously. There was no effect of IH on normoxic oxygen saturation during exercise, but IH caused a significant increase in saturation during hypoxic exercise. This increase in saturation was not associated with a concomitant increase in minute ventilation, so the reasons for this finding are unclear. Two potential explanations (decreased oxygen consumption, and improved oxygen delivery) are presented. Several studies7 10 23 have shown a decreased oxygen cost during submaximal running following IH with no change in exercise minute ventilation. This decreased oxygen consumption for a given workload has been attributed to improved efficiency. Potential mechanisms for enhanced efficiency include a decreased cost of breathing, greater carbohydrate utilisation for oxidative phosphorylation, or reduced consumption of adenosine triphosphate by the muscle10 23. A similar increase in exercise efficiency in the present study could explain the increased arterial oxygen saturation. Secondly, if the pattern of ventilation had changed, such that tidal volume was preferentially increased, improving alveolar ventilation, saturation would be increased without an overall increase in minute ventilation. The increased 88 hypoxic drive to ventilation could have led to a preferential increase in tidal volume thus reducing the physiological dead space and enhancing oxygen saturation. IH could also have improved arterial oxygenation with no change in ventilation through beneficial effects on diffusion capacity, lung ventilation/perfusion or a decrease in shunt (although these changes following IH have not been studied). In summary, several possible mechanisms could explain all or part of the improved saturation following the SDIH protocol; a more in-depth assessment of oxygen consumption, respiratory parameters and arterial blood gases would be required to better characterise this finding. Maximal Exercise Test When the data for both protocols was assessed, there were no significant differences in peak wattage, peak ventilation or peak oxygen consumption during a normoxic graded exercise test. From previous work, the effect of IH on maximal performance is unclear. Studies that have looked at maximal exercise performance in normoxia following IH have failed to show a significant increase in peak power output9 13 24 25, except with concurrent exercise training26. This study is the first to examine the effect of IH on peak exercise performance in hypoxia. Conceivably, IH could have improved oxygen delivery during hypoxic exercise, either through alterations in mitochondrial activity, muscle buffering capacity or oxygen delivery, but the current study provided no indication of such an improvement. 89 Unfortunately, due to technical constraints, the sample size for peak Vo2 determination was only 5. No significant changes in Vo2 were evident. A larger sample size would be necessary to confidently determine the effects of IH on peak oxygen consumption during exercise. SDIH vs. LDIH There were no significant differences among any of the measured variables between the two IH protocols in the current study. We had hypothesised that the SDIH protocol, which seemed to more profoundly augment carotid sinus nerve activity in animals27, may have had a similar effect in humans (observable as an increase in ventilatory response to hypoxia). Intermittent hypoxia appeared to enhance the hypoxic drive to breathe during submaximal exercise, but there was no differential benefit of SDIH over LDIH. This lack of benefit of SDIH over LDIH parallels the results found in the resting measures discussed in Chapter 3. Although the current study had no effect on submaximal minute ventilation, one could argue that if there had been a more profound augmentation of hypoxic ventilatory response as in the "live-high, train low" protocols, a difference between the two protocols might have become be evident. Unfortunately, the protocols that seem to have some effect on exercising ventilatory parameters (such as that of Townsend et al.11) are not conducive to SDIH, because tents and chambers need such a long time to establish hypoxia (30 minutes to 2 hours). 90 Limitations Due to subject fatigue and technical difficulties, the sample size was very limited for the final part of the graded exercise test. With such a low sample size (n = 5), a very large difference (or low variability) in the measured values would need to be present to be detectable. A rest period before the graded exercise test would give the subjects a short recovery period prior to attempting the graded exercise test. The disadvantage of this modification is that the exercise testing sessions for the subjects were already extremely long and this modification would lengthen it by approximately 20 minutes. With the current setup, breath-by-breath data was collected during the submaximal exercise bouts, and mixed gases were collected for the graded exercise testing. Using a parallel setup, it would be possible to collect both types of data throughout the entire test. Such a paradigm would provide valuable information about submaximal exercise efficiency. 91 CONCLUSIONS: Following two different 7-day IH protocols administered to subjects in a crossover fashion there was a slight increase submaximal oxygen saturation during hypoxic exercise. There were no changes in submaximal exercise ventilation in hypoxia or normoxia. Peak exercise ventilatory parameters and peak power were unchanged by IH. The response to hyperoxia during exercise was augmented by IH whereas the response to hypercapnia was unchanged by IH. No differences occurred between the SDIH and LDIH protocols in terms of respiratory response during exercise. Thus, although an augmentation of response to hyperoxia occurred following IH, it was not translated into an increase in minute ventilation during normoxic or hypoxic exercise. 92 REFERENCES 1. Shephard RJ. PAR-Q, Canadian Home Fitness Test and exercise screening alternatives. Sports Med 1988;5(3): 185-95. 2. Thomas S, Reading J, Shephard RJ. Revision of the Physical Activity Readiness Questionnaire (PAR-Q). Can J Sport Sci 1992; 17(4):338-45. 3. Sporer B, Koehle MS, Hodges ANH, Lane K, McKenzie DC. The pattern of arterial desaturation in exercise at various simulated altitudes. In Preparation. 4. Cooper T. Peripheral Chemoresponsiveness and Exercise Induced Arterial Hypoxemia in Highly Trained Endurance Athletes. [Master's Thesis]. University of British Columbia, 1993. 5. Fukuoka Y, Endo M, Oishi Y, Ikegami H. Chemoreflex drive and the dynamics of ventilation and gas exchange during exercise at hypoxia. Am J Respir Crit Care Med 2003; 168(9): 1115-22. 6. Katayama K, Sato Y, Shima N, Qiu JC, Ishida K, Mori S, et al. Enhanced chemosensitivity after intermittent hypoxic exposure does not affect exercise ventilation at sea level. Eur J Appl Physiol 2002;87(2): 187-91. 7. Katayama K, Matsuo H, Ishida K, Mori S, Miyamura M. Intermittent hypoxia improves endurance performance and submaximal exercise efficiency. High Alt Med Biol 2003;4(3):291-304. 8. Katayama K, Sato K, Matsuo H, Ishida K, Iwasaki K, Miyamura M. Effect of intermittent hypoxia on oxygen uptake during submaximal exercise in endurance athletes. Eur J Appl Physiol 2004;92(1-2):75-83. 9. Foster GE, McKenzie DC, Sheel AW. Effects of enhanced human chemosensitivity on ventilatory responses to exercise. Exp Physiol 2006;91(1):221-8. 10. Gore CJ, Hahn AG, Aughey RJ, Martin DT, Ashenden MJ, Clark SA, et al. Live high:train low increases muscle buffer capacity and submaximal cycling efficiency. Acta Physiol Scand 2001 ;173(3):275-86. 11. Townsend NE, Gore CJ, Hahn AG, Aughey RJ, Clark SA, Kinsman TA, et al. Hypoxic ventilatory response is correlated with increased submaximal exercise ventilation after live high, train low. Eur J Appl Physiol 2005;94(1-2):207-15. 93 12. West JB, Peters RM, Jr., Aksnes G, Maret KH, Milledge JS, Schoene RB. Nocturnal periodic breathing at altitudes of 6,300 and 8,050 m. J Appl Physiol 1986;61(1):280-7. 13. Katayama K, Sato Y, Morotome Y, Shima N, Ishida K, Mori S, et al. Intermittent hypoxia increases ventilation and Sa02) during hypoxic exercise and hypoxic chemosensitivity. J Appl Physiol 2001;90(4): 1431-40. 14. Loeppky JA, Icenogle M, Scotto P, Robergs R, Hinghofer-Szalkay H, Roach RC. Ventilation during simulated altitude, normobaric hypoxia and normoxic hypobaria. Respir Physiol 1997;107(3):231-9. 15. Tucker A, Reeves JT, Robertshaw D, Grover RF. Cardiopulmonary response to acute altitude exposure: water loading and denitrogenation. Respir Physiol 1983;54(3):363-80. 16. Powell FL, Milsom WK, Mitchell GS. Time domains of the hypoxic ventilatory response. Respir Physiol 1998;112(2):123-34. 17. Ling L, Fuller DD, Bach KB, Kinkead R, Olson EB, Jr., Mitchell GS. Chronic intermittent hypoxia elicits serotonin-dependent plasticity in the central neural control of breathing. 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Improved running economy in elite runners after 20 days of simulated moderate-altitude exposure. JApp/P/jys/o/2004;96(3):931-7. 24. Katayama K, Shima N, Sato Y, Qiu JC, Ishida K, Mori S, et al. Effect of intermittent hypoxia on cardiovascular adaptations and response to progressive hypoxia in humans. High Alt Med Biol 2001 ;2(4):501-8. 25. Rodriguez FA, Casas H, Casas M, Pages T, Rama R, Ricart A, et al. Intermittent hypobaric hypoxia stimulates erythropoiesis and improves aerobic capacity. Med Sci Sports Exerc 1999;31(2):264-8. 26. Katayama K, Sato Y, Ishida K, Mori S, Miyamura M. The effects of intermittent exposure to hypoxia during endurance exercise training on the ventilatory responses to hypoxia and hypercapnia in humans. Eur J Appl Physiol Occup Physiol 1998;78(3): 189-94. 27. Peng YJ, Prabhakar NR. Effect of two paradigms of chronic intermittent hypoxia on carotid body sensory activity. J Appl Physiol 2004,96(3):1236-42; discussion 1196. 95 CONCLUSIONS: 96 Intermittent hypoxic exposures increase the ventilatory response to hypoxia at rest. IH has the potential for applied and clinical benefits, including improving exercise performance1, reducing cardiac ischaemia2 and it may have a role in improving ventilation in spinal cord transection patients3. Many different protocol intensities, durations and duty cycles have been studied, but as yet the optimal IH protocol for augmenting HVR in humans is not yet known. The purpose of the study was to compare the effects of two different types of intermittent hypoxia protocols on respiratory chemoresponse and to examine the relationship between carbon dioxide and oxygen sensitivity during rest and exercise. Furthermore, HVR was assessed daily to follow the pattern of HVR augmentation during the course of IH. To determine whether repeated measurement of HVR caused a confounding co-intervention, repeatability study was performed. This study demonstrated: 1) acceptable values for coefficient of variation for HVR and 2) no apparent IH stimulus from daily measurements of HVR. Two types of intermittent hypoxia: short-duration intermittent hypoxia (SDIH) and long duration intermittent hypoxia (LDIH) were assessed in the second phase of the project. Ten male subjects underwent two seven-day intermittent hypoxic training protocols while being assessed for a battery of measurements of resting and exercise ventilatory control. As anticipated, following the IH, HVR was augmented and CO2 threshold was decreased (in hypoxia and hyperoxia). HCVR was also increased by IH. These resting alterations in chemoreflex control remained different from baseline 97 at 7 days post-IH. Intermittent hypoxia had no effect on exercise minute ventilation, but increased the response to hyperoxic challenge. Also during submaximal exercise, there was a slight increase in submaximal O2 saturation (by oximetry). Peak exercise values were unchanged by intermittent hypoxia. It was initially hypothesised that SDIH would cause more profound changes in the measured variables than LDIH. However, the effects of SDIH and LDIH were similar throughout, with no significant differences for any of the measurements. Significance This research comprehensively compares SDIH and LDIH, demonstrating no significant differences between the two protocols. It is the first research to examine HVR, HCVR and the modified Read rebreathing techniques in the same subjects following the same intervention. It confirms that IH both augments HVR and lowers the CO2 threshold, but that there were no correlations between these two parameters. It also highlights (and discusses potential mechanisms for) the discrepancy between CO2 sensitivity as measured by HCVR and by the modified rebreathing method. Furthermore, this work comprises the most comprehensive assessment of resting and exercising chemosensitivity in subjects undergoing IH. Previous work examining the relationship between resting chemosensitivity and exercise control of breathing have not employed specific chemosensitivity tests or have focussed on peak exercise ventilation. For the IH protocols tested, augmentation of resting hypoxic ventilatory response resulted in an increase in 98 exercising chemosensitivity. This alteration did not affect the minute ventilation or the change in exercise ventilation between normoxia or hypoxia, indicating that its effect on the control of breathing during exercise was modest at best. Further analysis is required to assess the changes in respiratory pattern (such as tidal volume and frequency of breathing) that may potentially occur following IH. An increased tidal volume during submaximal ventilation could potentially explain the enhanced saturation during hypoxic exercise without an increase in minute ventilation. The current experimental design with it's systematic assessment of resting and exercising chemoresponse in a crossover controlled fashion is the most sophisticated comparison of SDIH and LDIH on the control of breathing in humans. The results fail to support any benefit of SDIH over LDIH on the augmentation of chemoresponse in humans. This information lends credence to the concept that the normoxic-hypoxic transients that are more plentiful in SDIH are not instrumental to the augmentation of ventilation in humans. Limitations & Future Research Ventilatory measures during wakefulness have a high degree of variation. The repeatability study demonstrated that the technique for measurement of HVR used in the lab shows a sizeable coefficient of variation (27%), but one which is comparable to the lowest previously reported values. Although the other testing methods used in this research have not been assessed in terms of their repeatability, they likely have significant coefficients of variation as well. It is 99 therefore possible that changes occurred in the chemosensitivity to hypoxia or hypercapnia that were not detected due to this variation. Larger alterations in resting chemoreceptor control may be needed to induce detectable changes in the ventilatory control of breathing during exercise. A similarly extensive assessment of resting and exercising chemoresponse, but with a "live-high, train-low" protocol would provide valuable insight not only into the effects of IH, but also on the control of breathing during exercise in humans. Potential mechanisms for the increased saturation during submaximal exercise following IH include an increase in alveolar ventilation or a decrease in oxygen consumption (improved efficiency). One could derive approximate values for oxygen consumption by integrating the breath-by-breath data collected during this study. Ultimately, a more detailed assessment of gas exchange, using arterial blood gases and a combination of both breath-by-breath and mixing chamber analysis during submaximal exercise would provide more definitive answers regarding the effects of IH on exercise efficiency. The differences between normobaric and hypobaric IH remain unclear. It is tempting to equate the hypoxia from a reduced FT02 in normobaria with a constant F|02 in hypobaria. The two conditions are not identical and may have differential effects on fluid status, baroreceptor tone and other physiological processes that affect the response to hypoxia. A head-to-head trial comparing a hypobaric IH protocol with its normobaric equivalent, although technically challenging, would provide valuable insight into these differences. 100 The importance of sleep during IH is also not understood. IH protocols that involve sleeping in hypoxia4 cause greater increases in HVR than when the subjects are awake5 6. It is unclear whether this due to the duration of the hypoxic exposure or the state of wakefulness. A comparison of two IH protocols with a consistent exposure duration, and F|02, but with subjects either awake or asleep would provide interesting insight. Summary IH has many potential applied and clinical applications. A thorough understanding of its mechanism of action and optimal dosages would be beneficial to properly tailor IH for its intended use. From this work it appears that a 7-day course of poikilocapnic IH causes 1) an increase in HVR that occurs rapidly in the first four days of exposure, 2) a left-shift in the CO2 threshold in both hypoxic and hyperoxic conditions, 3) a concomitant increase in the ventilatory response to hyperoxic challenge during exercise 4) all changes are essentially equivalent following an SDIH or an LDIH protocol. This work raises many important questions that provide opportunity for further study. 101 REFERENCES: 1. Katayama K, Matsuo H, Ishida K, Mori S, Miyamura M. Intermittent hypoxia improves endurance performance and submaximal exercise efficiency. High Alt Med Biol 2003;4(3):291-304. 2. Zong P, Setty S, Sun W, Martinez R, Tune JD, Ehrenburg IV, et al. Intermittent hypoxic training protects canine myocardium from infarction. Exp Biol Med (Maywood) 2004;229(8):806-12. 3. Golder FJ, Mitchell GS. Spinal synaptic enhancement with acute intermittent hypoxia improves respiratory function after chronic cervical spinal cord injury. J Neurosci 2005;25(11):2925-32. 4. Townsend NE, Gore CJ, Hahn AG, Aughey RJ, Clark SA, Kinsman TA, et al. Hypoxic ventilatory response is correlated with increased submaximal exercise ventilation after live high, train low. Eur J Appl Physiol 2005;94(1-2):207-15. 5. Foster GE, McKenzie DC, Milsom WK, Sheel AW. Effects of two protocols of intermittent hypoxia on human ventilatory, cardiovascular and cerebral responses to hypoxia. J Physiol 2005,567(Pt 2):689-99. 6. Katayama K, Fujita H, Sato K, Ishida K, Iwasaki K, Miyamura M. Effect of a repeated series of intermittent hypoxic exposures on ventilatory response in humans. High Alt Med Biol 2005;6(1 ):50-9. 102 APPENDIX I: Certificates of Ethical Review The University of British Columbia Office of Research Services and Administration Clinical Research Ethics Board Certificate of Expedited Approval UBC Campus CO-WVESTOATORS: Guenette, Jordan,; Koehle, Michael, Human Kinetics; McKenzie, Donald, Human Kinetics-bporer, Benjamin, Human Kinetics ' Unfunded Research APPROVAL DATE OCT 01 2003 CERTIFICATION: In iwpKt of clinical trial*: • -I Consent form version 1.1 dd 4 September 2003; posters, protocol 1. The membership of this Research Ethics Board complies with the membership requirements for Research Ethics Boards defined in Division 5 of Vie Food and Drug Regulations. 2. The Research Ethics Board carries out Hs functions in a manner consistent with Good Clinical Practices. 3. This Research Ethics Board has reviewed and approved the clinical trial protocol and informed consent form for the trial which is to be conducted by the qualified investigator named above at the specified clinical trial site. This approval and the views of the this Research Ethics Board have been documented In writing. The documentation included for the above-named project has been reviewed by the Chair of the UBC CREB, and the research study, as presented In the documentation, was found to be acceptable on ethical grounds for research involving human subjects and was approved by the UBC CREB. The CREB approval for this study expires one year from the approval date. Approval of the Clinical Research Ethics Board by one of: Dr. P. Loewen, Chair Dr. A. Gagnon, Associate Chair Dr. J. McCormack, Associate Chair 104 The University of British Columbia Office of Research Services. Clinical Research Ethics Board - Room 210. S2S West 1C* Avenue. Vancouver, BC Vf Z 1L3 Certificate of Expedited Approvai Clinical Research Eihics Board Official Notification DCl'tKtUtM McKenzie. DC mstfij-iMH'S' WMttfi- Hfsj-jun-* Will *# • '\ Family Practice l" C04-0402 UBC Campus UMfcVfc51K~M-M!f *~~~~~~~~_ —^—______ , _ Guenette. Jordan.: Hughes. Bevan,. Koehle. Michael. Human Kinetics: Lusina. Sarah. Human Kinetics: Miisom. William. Zoology. Sheel William. Human Kinetics SfOhSQRlhO ACef«U>£S ----------------------------------------Natural Science Engineering Research Council wu Intermittent Hypoxia and the Chemoreflex Control of Ventilation K--UH.U *.t IN 'tf* WWi 11 Aug 2004 1 Protocol: Consent version 1.1 dated 2" My 2004: Advertisement: PAR-Q Qjies&onnaire In r*«p*ct of clinical trials: 1 The membership of this Research Etftci Board complies mth the membership requirements for Research Ethics Boards defined in Dwswn 5 of the Food and Drug Regulations 2. The Research Ethics Board cames out its functions m a manner consistent with Good Clinical Practices. 3. This Research Ethics Board has reviewed and approved She clinical trial protocol and informed consent form for the tnal *hteft is to be conducted by the qualified investigator named above at the specified clinical trial site This approvai and the views of the this Research Ethics Board have been documented in writing The doc-mentation included fortne above-named project has been reviewed by the Chair of the JBC CREB and the research study, as preserteo* in trie documentation, was found to be acceptable on ethical grounds for research invcvmg human subjects and was approved by the UBC CREB. The CREB approval for this study expires one year from the approval date. Approval of the Clinical Research Ethics Board by one of Dr P. Loewen. Chair Dr. A. Gaenon, Associate Chair Dr. J. McCormack. Associate Chair UBC The University of British Columbia Office of Researcn Services. Ciimcal Research Ethics Board - Room 210 S23 'A'est 10* Avenue. Vancouver, BC VEZ 1LS 105 [UBC w The University of British Columbia Office of Research Services, Clinical Research Ethics Board - Room 210 328 West 10* Avenue, Vancouver. BC VS2 1L3 Certificate of Expedited Approval: Renewal Clinical Research Ethics Board Official Notification lnwin-A. wtsWo* -mnrrstm McKenzie. D.C. C04-0402 UBC Campus <.04hVt£tO*IO>(S ——— Guenette. Jordan.: Hughes. Bevan.: Koehle. Michael. Human Kinencs; Lu5ina. Sarah. Human Kinetics: Milsom. William. Zoology. Sheet. William. Human Kinetics Natural Science Engineering Research Council Intermittent Hypoxia and the Chemoreflex Control of Ventilation 11HM imMi:— tMN'-'UtrHf 19 July 2005 1 hi raepect or clinical trtaia: 1. The membership of this Research Ethics Board complies with the membership requirements for Research Ethics Boards defined in Division 5 cf the Food and Drug Regulations 2 The Research Ethics Board cames out ns functions m a manner consistent frith Good Clinical Practices. 3. This Research Ethics Board has reviewed and approved the dinical tnal protocol and informed consent form hr the trial mlvch is to be conducted by the qualified investigator named above at the specified c'm<:ai trial site This approval and the views of this Research Ethics Board have been documented in t/rritmg The Chair of the UBC Clinical Research Ethics Board has reviewed the documentation for the above named project. The research study, as presented in the documentation, was found to be acceptable on ethical grounds for research involving human subjects and was approved for renewai by the UBC Clinical Research Ethics Board The CREB approval for renewal of this study expires one year from the date of renewal. Approval of the Clinical Reseanh Ethics Board by one of Dr Gail BellwardL Chair Dr. James McCormack. Associate Chair 106 APPENDIX II: Informed Consent Forms capable of inducing haematological changes, such as increases in the ability of the blood to carry oxygen. The ideal protocol is not yet known. In animals, multiple short bouts (less than 5 minutes) each day has been shown superior to single long daily bouts. This has not been examined in humans. Likewise the ideal length of an IHT protocol is also unknown. This study will attempt to answer these questions. Purpose: The purpose of this study is to investigate the breathing response to hypoxia over seven daily hypoxic ventilatory response tests. Procedures: All subjects recruited for the study will be normal healthy male volunteers, between 18-40 years of age. All subjects will be non-smoking, have normal pulmonary function and free of any history or symptoms of cardiopulmonary disease including exercise-induced asthma. Subjects will not have had any significant exposure to altitude or hypoxia in the preceding four weeks. Each subject will undergo a standardized screening history (Physical Activity Readiness Questionnaire; PAR-Q). If you consent to become a subject in this study you will be asked to participate in nineteen data collection test days. The session will take place at the Health and Integrated Physiology Laboratory at the Osborne Centre (Unit 2, Room 202) on the University of British Columbia campus. The study will require approximately thirty-four (34) hours of your time. We will schedule your testing sessions to be most convenient for you. On the first day, your height and weight will be measured. You will then undergo a simple, non-invasive breathing test to ensure that you do not have any obstructive lung disease (i.e., asthma). This requires you to breathe deeply and exhale quickly through a mouthpiece. You will then be required to lie comfortably on a bed in which you will breath through a two-way valve so that expired gases and flow can be monitored. A small plastic clip will slip onto your fingertip. This will permit us to measure the amount of oxygen in your blood. After 10 minutes of breathing normal air, experimenters will slowly and progressively add nitrogen gas to the air you are breathing. We will measure the amount that your breathing (rate and depth) increases in response to this. The test will stop once your blood oxygen saturation level reaches 80%. This experiment will simulate high altitude exposure and will take approximately 15 minutes. This is the hypoxic ventilatory response (HVR) test. You will then perform a similar test where you breathe from a large bag while resting. We will control the concentration of gases in the bag. Gradually the carbon dioxide in the bag will accumulate, and you will breathe more and more. We will stop the test once it has become too uncomfortable or the amount of carbon dioxide in the bag reaches a pre-determined amount (60 mmHg). This is the hypercapnic ventilatory response test (HCVR). 109 The next test is the maximal oxygen uptake test. This is a test where you ride a stationary bicycle while wearing a mask to collect the gas that you breath out. The resistance on the bicycle gets higher and higher until you can no longer continue. This test determines aerobic fitness. On your second visit, you will repeat the HVR test and the HCVR test. You will also complete the multi-stage exercise test. During this test, you will be exercising on a bicycle at two relatively low resistances. You will first exercise breathing room air, and then secondly while breathing hypoxic air. You will finish the exercise test by performing another maximal oxygen uptake test (while breathing the hypoxic gas). All your pre-testing is complete. You will then start your intermittent hypoxic training (IHT). This will last for 7 days. You will do two different protocols, and they will each last seven days. Each day you will come to the lab, and breath a gas mixture while relaxing, watching movies, reading or working quietly. Each time you come in you will also do an HVR test. At the end of each 7-day IHT session we will repeat the HVR, HCVR and multi-stage exercise test. You will do each IHT programme at least 2 weeks apart. One week after each IHT programme, you will return to the lab and HVR and HCVR will be re-measured. Risks: There are no significant risks associated with a short exposure to simulated altitude (approximately 20,000 feet). A physician (Dr. Koehle or Dr. Hughes) will be present at all testing sessions, if you feel any discomfort, or have any concerns, you will be attended to immediately. Some people find it feels a little uncomfortable when they are breathing hypoxic air. The maximal oxygen uptake test has a small chance of adverse effects, such as vomiting (5%), abnormal blood pressure (less than 1%). fainting (less than 1%), disorders of the heartbeat (less than 0.1%). and very rare instances of heart attack (less than 0.001%). All procedures used in this study have been previously performed in our laboratory without incident. Benefits: By participating in the study, the subjects will enhance the understanding of the effects of hypoxia on the control of breathing; this knowledge will be used to further our understanding of the safety of diving in the asthmatic population. Furthermore, you will receive a maximal oxygen uptake test (V02max test) and two courses of intermittent hypoxic training at no charge. After completion of the study, you will receive an honorarium of one hundred dollars. Confidentiality: Your rights to privacy are protected by the Freedom of Information and Protection of Privacy Act of British Columbia This Act lays down rules for the collection, protection, and retention of your personal information by public bodies, such as the University of British Columbia and its affiliated teaching hospitals. Further details about this Act are available upon request. Your confidentiality will be respected. No information that 110 discloses your identity will be released or published without your specific consent to the disclosure. However, research records and medical records identifying you may be inspected in the presence of the Investigator or his or her designate by representatives of the UBC Research Ethics Board for the purpose of monitoring the research. However, no records which identify you by name or initials will be allowed to leave the Investigators' offices. You are encouraged to ask for an explanation or clarification of any of the procedures or other aspects of this study before signing this consent from or at any time during your participation in the study. YOU MAY DECLINE TO ENTER THIS STUDY OR WITHDRAW FROM THE EXPERIMENT AT ANY TIME. If you have any concerns or questions about your rights or experience as a research subject, you may contact the Research Subject Information Line in the UBC Office of Research Services at (604) 822-8598. Consent: In signing this form you are consenting to participate in this research project and acknowledge receipt of a copy of this form. Signing this consent form in no way limits your legal rights against the sponsor, investigators, or anyone else. Signature of Participant Date Printed Name of Participant Signature of Witness Date Printed Name of Witness Signature of Investigator Date Printed Name of Investigator 111 (less than 5 minutes) each day has been shown superior to single long daily bouts. This has not been examined in humans. Likewise the ideal length of an IHT protocol is also unknown. This study will attempt to answer these questions. Purpose: The purpose of this study is to investigate the breathing response to hypoxia over seven daily hypoxic ventilatory response tests. Procedures: All subjects recruited for the study will be normal healthy male volunteers, between 18-40 years of age. All subjects will be non-smoking, have normal pulmonary function and free of any history or symptoms of cardiopulmonary disease including exercise-induced asthma. Subjects will not have had any significant exposure to altitude or hypoxia in the preceding four weeks. Each subject will undergo a standardized screeriing history (Physical Activity Readiness Questionnaire; PAR-Q). If you consent to become a subject in this study you will be asked to participate in nineteen data collection test days. The session will take place at the Health and Integrated Physiology Laboratory at the Osborne Centre (Unit 2, Room 202) on the University of British Columbia campus. The study will require approximately thirty-four (34) hours of your time. We will schedule your testing sessions to be most convenient for you. On the first day, your height and weight will be measured. You will then undergo a simple, non-invasive breathing test to ensure that you do not have any obstructive lung disease (i.e., asthma). This requires you to breathe deeply and exhale quickly through a mouthpiece. You will then be required to lie comfortably on a bed in which you will breath through a two-way valve so that expired gases and flow can be monitored. A small plastic clip will slip onto your fingertip. This will permit us to measure the amount of oxygen in your blood. After 10 minutes of breathing normal air, experimenters will slowly and progressively add nitrogen gas to the air you are breathing. We will measure the amount that your breathing (rate and depth) increases in response to this. The test will stop once your blood oxygen saturation level reaches 80%. This experiment will simulate high altitude exposure and will take approximately 15 minutes. This is the hypoxic ventilatory response (HVR) test. You will then perform a similar test where you breathe from a large bag while resting. We will control the concentration of gases in the bag. Gradually the carbon dioxide in the bag will accumulate, and you will breathe more and more. We will stop the test once it has become too uncomfortable or the amount of carbon dioxide in the bag reaches a pre-determined amount (60 mmHg). This is the hypercapnic ventilatory response test (HCVR). 113 The next test is the maximal oxygen uptake test. This is a test where you ride a stationary bicycle while wearing a mask to collect the gas that you breath out. The resistance on the bicycle gets higher and higher until you can no longer continue. This test determines aerobic fitness. On your second visit, you will repeat the HVR test and the HCVR test. You will also complete the multi-stage exercise test. During this test, you will be exercising on a bicycle at two relatively low resistances. You will first exercise breathing room air, and then secondly while breathing hypoxic air. You will finish the exercise test by performing another maximal oxygen uptake test (while breathing the hypoxic gas). All your pre-testing is complete. You will then start your intermittent hypoxic training (IHT). This will last for 7 days. You will do two different protocols, and they will each last seven days. Each day you will come to the lab, and breath a gas mixture while relaxing, watching movies, reading or working quietly. Each time you come in you will also do an HVR test. At the end of each 7-day IHT session we will repeat the HVR, HCVR and multi-stage exercise test. You will do eachTHT programme at least 2 weeks apart. One week after each IHT programme, you will return to the lab and HVR and HCVR will be re-measured. , Risks: There are no significant risks associated with brief mild hypoxia exposure (approximately 20,000 feet). A physician (Dr. Koehle or Dr. Hughes) will be present at all testing sessions, if you feel any discomfort, or have any concerns, you will be attended to immediately. Some people find it feels a little uncomfortable when they are breathing hypoxic air. The maximal oxygen uptake test has a small chance of adverse effects, such as vomiting (5%), abnormal blood pressure (<1%), fainting (<1%), disorders of the heartbeat (<0.1%), and very rare instances of heart attack (<0.001%). All procedures used in this study have been previously performed in our laboratory without incident. Benefits: By participating in the study, the subjects will enhance the understanding of the effects of hypoxia on the control of breathing; this knowledge will be used to further our understanding of the safety of diving in the asthmatic population. Furthermore, you will receive a maximal oxygen uptake test (V02max test) and two courses of intermittent hypoxic training at no charge. After completion of the study, you will receive an honorarium of one hundred dollars. Confidentiality: Your rights to privacy are protected by the Freedom of Information and Protection of Privacy Act of British Columbia, This Act lays down rules for the collection, protection, and retention of your personal information by public bodies, such as the University of British Columbia and its affiliated teaching hospitals. Further details about this Act are available upon request. Your confidentiality will be respected. No information that discloses your identity will be released or published without your specific consent to the disclosure. However, research records and medical records identifying you may be 114 inspected in the presence of the Investigator or his or her designate by representatives of the UBC Research Ethics Board for the purpose of monitoring the research. However, no records which identify you by name or initials will be allowed to leave the Investigators' offices. You are encouraged to ask for an explanation or clarification of any of the procedures or other aspects of this study before signing this consent from or at any time during your participation in the study. YOU MAY DECLINE TO ENTER THIS STUDY OR WITHDRAW FROM THE EXPERIMENT AT ANY TIME. If you have any concerns or questions about your rights or experience as a research subject, you may contact the Research Subject Information Line in the UBC Office of Research Services at (604) 822-8598. Consent: In signing this form you are consenting to participate in this research project and acknowledge receipt of a copy of this form. Signing this consent form in no way limits your legal rights against the sponsor, investigators, or anyone else. Signature of Participant Date Printed Name of Participant Signature of Witness Date Printed Name of Witness Signature of Investigator Date Printed Name of Investigator 115 APPENDIX III: Physical Activity Readiness Questionnaire 116 frevfced&B) PAR-Q & YOU (A Questionnaire for People Aged 15 te 69) Heguter pjipical MES?/ e fun ind heafihy, and irtoeasmarr snore psopte are stating to beams mere ac.iie srvery day Being more aaae is very safe ior most paaple. :-to,»?i-e-r, some people should check sr-h tSieir doctor before they sart bacsimng irrudi more physicElrjr active. E ira i*9 farming to secoms much mors pbysxaDy ear/9 Chan joa ere now, start by sewing tie seven questions in the ben beta* B you are bsraean ths a§asof 1 Sand 69. the SW-Q w3l tell jou if ymrshaiM died wife your doaor beta you start If ytraswovar&'SyBarsol age, and you are not used to being wry scwe, died wilh your doom Common tense is your best guide whan you answer these qussocre. Please read ths questions CBrefuuy aid answer each one honesty: check YES cr KO. res NO • • t. • • 2. • • 3, • • 4. • • S. • • e. • • 7. Has year doctor ever said that yen have a heart condition and that yea should only de physical activity recommended by a doctor? De yea feel pain in year chest when yea do physical activity? ID the past month, have yoo had chest pain when yen sere net doing physical activity? De yea lose your balance became et dizziness or de yea ever lese consciousness? De yea have a bone or joint problem (far eiample, bach, fcnee or hip) thai could be made worse by a change in year physical activity? Is your doctor currently preicrrbbo drags (fei example, water pills) for your bleed pressure er heart con dition? De yen know ef aw ether reason why yen should net de physical activity? If you answered YES to one or more questi Talk Atth pur doctor ay pfcens crfe parson 3EF0FE pu start becctring such men gkj/zto&f BTDW cr EcFCRE yoy hstti a Stress appraisal. Isti ycur dbctsr about the PMHJ 2nd which quaciraB ycu are«red YES. • Ycu rray he aifo to do any xzjri.ty you want— as Inng ss ycu siart sfcwty UP ffraduaf^ Cr, yirj may need :o reSTirt pur adidte; to dtcse o^sch S7e safe fer you. Talk nrtrt your doctor stout the khds cf tctrrtins pu wish to por^cbats in and icilc* holier actfea. • nrtd out which tntmtifuijr prograres are safe and IndbAil fcr yoa_ NO to all questions OSLAT BECOMING MUCH ROBE ACTIVE: - IJ yaa a» rwc fee3ag •sfl bocaiaff cf atBitpcraryanssssuchas a cok) cr s fcwr- mst urei pu fed better; ar * if pu are or may be pregnane-tall tu your doctcr barcre pu stan baemthg rocrg aicu«. tf you answered HO fecnststJ/ ta all flAft< gjsstixsv pu can be raasonanV aire that ycu can: • scan iwccrrinj such nxra cfcyscaly actna - begin SIS-AI/ and inftf up gradual}/ Ihc b the safest and eases: way to ga • take part h a fJteess appraisal— ths ts an excellent way to datorafee pur basic £biess so ftalpacanptrnthst^wayfarpu to he aAM*}/ fto alst highly /•rafrrnsjiCBd lha* yaa haws ycur Stoxi pressure evaluated. 2 pur reading is ever 144f94, tdk with pur &ctef bsfcre ycu start becKnirg micfi room physicafy iHrrc. PIEASENOTE: IF pur haarT". dongas sc that ycrj then ans-rvcr YE5 :a any of th? above qnKfanns, td\ yxr£rneu cr h=aeh prtricscnai. Ask 'tvhethsr pu shoJd change year physkal activity plan. He changes permitted. Toil are encouraged te photocopy the PAR-Q bat only if yea use (he entire form, 1 nave read, understood and completed this questionnaire. Any cntessceis I had wars anseered to UI sabsfactim.* mm 99K?.IE am Hate: Tbb physical activity etc ex once is valid fnor a Biaiisntiaa of 12 maatbs from tlie date it b completed and becomes invalid if your condition cfaages so tliat yon would answer TES to any of the seven cjuestknts. ® CansdzD SccTirty fcr Ectyose Ptyzkhq$ Suppcrledtr^ Canada C^iriada ccfitnueQ >cm other sirfs... 117 APPENDIX IV: Data Tables Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 14 Subject HVR R2 HVR R2 HVR R2 HVR R2 HVR R2 HVR R2 HVR R2 HVR R2 HVR R2 1 0.24 0.32 0.36 0.66 0.41 0.43 0.56 0.63 0.36 0.77 0.41 0.51 0.41 0.61 0.33 0.49 0.39 0.59 2 0.43 0.83 0.26 0.65 0.25 0.67 0.62 0.88 0.75 0.83 0.35 0.69 0.59 0.89 0.66 0.84 0.71 0.48 3 0.22 0.35 0.30 0.45 0.62 0.64 0.54 0.58 0.41 0.57 0.43 0.48 0.68 0.59 0.73 0.58 0.56 0.50 4 0.24 0.41 0.33 0.57 0.28 0.40 0.42 0.66 0.46 0.43 0.72 0.91 0.52 0.74 0.49 0.71 0.47 0.78 5 0.67 0.59 0.84 0.63 0.97 0.32 0.82 0.81 1.11 0.82 0.63 0.67 0.79 0.63 0.65 0.75 0.82 0.69 6 0.46 0.35 0.46 0.33 0.47 0.66 0.41 0.38 0.45 0.60 0.61 0.61 0.68 0.69 0.60 0.64 0.58 0.55 7 1.01 0.82 1.22 0.78 2.20 0.90 1.75 0.92 1.53 0.87 1.06 0.93 1.06 0.94 1.14 0.81 0.58 0.87 8 0.29 0.72 0.25 0.74 0.42 0.64 0.51 0.54 0.30 0.82 0.35 0.55 0.39 0.46 0.36 0.55 0.35 0.74 9 0.57 0.37 0.98 0.69 0.87 0.58 1.28 0.73 1.13 0.65 1.24 0.63 0.84 0.81 1.41 0.79 0.87 0.84 10 0.56 0.80 0.53 0.79 0.67 0.82 0.54 0.86 0.72 0.83 0.89 0.80 0.70 0.67 0.62 0.90 0.54 0.79 Mean 0.47 0.5S 0.55 0.63 0.71 0.61 0.75 0.70 0.72 0.72 0.67 0.68 0.66 0.70 0.70 0.71 0.59 0.68 SD 0.25 0.22 0.34 0.15 0.57 0.18 0.43 0.17 0.41 0.15 0.31 0.16 0.20 0.15 0.34 0.13 0.17 0.14 Table 6.1: HVR results during the SDIH protocol. SD- standard deviation. HVR values are expressed in litresmin"1%Sa02'1 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 14 Subject HVR R2 HVR R2 HVR R2 HVR R2 HVR R2 HVR R2 HVR R2 HVR R2 HVR R2 1 0.261 0.461 0.484 0.634 0.455 0.508 0.807 0.545 0.488 0.703 0.778 0.749 0.824 0.592 0.734 0.661 0.651 0.473 2 0.414 0.748 0.298 0.513 0.408 0.716 0.485 0.697 0.477 0.814 0.477 0.793 1.111 0.637 0.694 0.884 0.580 .0.861 3 0.576 0.774 0.674 0.615 0.768 0.785 0.771 0.471 0.704 0.448 0.826 0.539 0.504 0.784 0.599 0.745 0.652 0.536 4 0.212 0.504 0.579 0.884 0.469 0.668 0.456 0.342 0.619 0.785 0.996 0.817 0.656 0.634 1.001 0.823 0.541 0.678 5 0.391 0.508 0.846 0.463 1.036 0.644 1.065 0.669 0.577 0.350 0.719 0.578 0.722 0.649 0.936 0.658 0.778 0.566 6 0.327 0.839 0.796 0.398 0.417 0.470 0.551 0.577 0.740 0.471 0.520 0.660 0.835 0.603 0.696 0.632 0.650 0.532 7 0.875 0.633 0.954 0.842 1.409 0.873 1.222 0.807 1.081 0.948 1.500 0.856 0.981 0.775 1.704 0.851 1.594 0.826 8 0.334 0.452 0.450 0.519 0.414 0.571 0.387 0.496 0.499 0.504 0.457 0.553 0.414 0.665 0.261 0.623 0.295 0.628 9 0.844 0.883 0.782 0.737 0.802 0.784 0.622 0.839 0.649 0.694 0.750 0.704 0.754 0.650 0.814 0.767 0.500 0.658 10 0.470 0.821 0.634 0.803 0.414 0.750 1.039 0.751 0.791 0.796 0.708 0.928 0.578 0.854 0.416 0.766 0.391 0.744 Mean 0.470 0.662 0.650 0.641 0.659 0.677 0.741 0.619 0.662 0.651 0.773 0.718 0.738 0.684 0.785 0.741 0.663 0.650 SD 0.229 0.170 0.202 0.169 0.343 0.130 0.289 0.160 0.183 0.196 0.306 0.134 0.213 0.088 0.391 0.094 0.356 0.129 able 6.2: HVR results during the LDIH protocol. SD- standard deviation. HVR values are expressed in litresmin ToSaCV1 SDIH Subject Pre- Post-7 days Post-1 1.57 2.63 2.64 2 3.26 3.27 2.41 3 2.49 2.59 2.77 4 4.34 7.52 5.78 5 2.67 3.06 3.78 6 1.40 1.28 1.80 7 5.86 3.97 5.08 8 4.93 4.59 5.22 9 3.27 4.32 3.00 10 2.07 5.23 3.68 Mean 3.19 3.85 3.62 Standard Deviation 1.47 1.73 1.34 Table 6.3: HCVR results during the LDIH and SDIH protocols. LDIH Subject Pre- Post-7 days Post-1 2.46 2.70 1.47 2 2.18 4.79 2.70 3 2.86 .2.57 3.21 4 1.05 3.34 4.12 5 3.95 6.29 6.17 6 2.86 2.63 2.36 7 3.37 5.77 4.73 8 4.43 4.77 5.94 9 2.07 2.32 2.52 10 3.13 5.69 6.92 Mean 2.84 4.09 4.01 Standard Deviation 0.97 1.54 1.86 HCVR values are expressed in litresmin~10/ommHg -1 SDIH Threshold Sensitivit y Subject Pre- Post-7 days Post- Pre- Post-7 days Post-1 44.12 42.20 42.66 2.48 1.56 3.49 2 45.23 42.59 42.72 3.06 4.17 3.30 3 44.22 41.53 47.16 2.61 1.73 2.32 4 47.30 42.43 43.15 4.68 4.10 4.40 5 45.11 41.93 44.86 3.71 2.91 3.38 6 48.92 45.78 48.89 1.72 1.64 2.31 7 45.76 45.22 48.56 2.82 3.14 4.54 8 43.44 42.24 43.66 5.21 4.25 4.82 9 44.26 43.13 44.03 2.49 2.78 1.91" 10 49.61 45.61 46.56 4.29 3.43 2.69 Mean 45.80 43.27 45.23 3.31 2.97 3.32 Standard Deviation 2.12 1.63 2.39 1.12 1.05 1.02 LDIH Threshold Sensitivity Subject Pre- Post-7 days Post- Pre- Post-7 days Post-1 47.91 41.84 43.02 2.31 2.48 3.02 2 47.15 41.64 43.35 4.28 6.59 4.85 3 44.46 43.05 43.05 2.05 1.78 2.37 4 49.07 47.55 48.45 3.84 3.29 4.86 5 44.11 43.06 44.05 3.50 4.34 4.09 6 46.81 46.70 47.60 3.38 2.81 2.68 7 45.21 43.37 45.98 4.27 2.12 2:81 8 42.06 42.58 42.97 5.75 3.67 3.91 9 43.75 44.02 42.33 1.98 2.16 1.94 10 49.31 45.41 46.22 4.15 3.07 5.10 Table 6.4: Hypoxic modified rebreathing method results during the LDIH and SDIH protocols. Threshold units mmHg. Sensitivity values are expressed in litresmin'1%mmHg"1 Mean 45.98 43.92 44.70 3.55 3.23 3.56 Standard Deviation 2.43 2.01 2.18 1.18 1.41 1.14 SDIH Threshold Sensitivity Subject Pre- Post-7 days Post- Pre- Post-7 days Post-1 39.73 37.86 38.75 4.70 5.01 4.54 2 40.00 37.96 41.35 4.37 5.82 6.40 3 40.54 40.94 42.25 4.94 5.19 5.68 4 44.16 38.02 41.40 5.00 4.80 5.75 5 37.75 39.33 39.54 4.17 4.60 4.85 6 46.58 41.77 41.77 2.60 2.80 3.90 7 44.35 40.30 42.93 4.40 6.55 7.46 8 36.29 37.85 37.64 7.56 7.51 6.11 9 40.71 38.93 39.83 5.40 5.48 3.80 10 43.82 41.15 42.34 4.93 3.29 8.98 Table 6.5: Hyperoxic modified rebrea LDIH Threshold Mean 41.39 39.41 40.78 4.81 5.11 5.75 Standard Deviation 3.24 1.52 1.74 1.23 1.39 1.61 Sensitivity Subject Pre- Post-7 days Post- Pre- Post-7 days Post-1 39.86 38.32 39.15 3.80 5.72 4.45 2 39.63 39.64 39.39 10.71 7.58 7.37 3 40.69 38.48 38.97 3.89 3.13 4.48 4 43.15 40.98 40.31 4.43 4.12 4.44 5 38.61 36.59 37.66 6.58 4.69 4.16 6 40.43 38.61 43.15 4.48 4.24 5.43 7 42.86 39.93 42.73 7.98 6.34 5.57 8 36.87 36.06 37.45 6.58 5.23 9.24 9 39.64 39.52 39.39 3.35 3.61 3.35 10 43.64 41.27 41.97 4.44 5.51 3.96 Mean 40.54 38.94 40.02 5.62 5.02 5.25 Standard Deviation 2.14 1.70 2.00 2.33 1.34 1.79 hing method results during the LDIH and SDIH protocols Threshold units mmHg. Sensitivity values are expressed in litresmin"1%mmHg -1 Mean Minute Ventilation Pre-IH Mean Minute Ventilation Post-IH SDIH Normox c Hypoxic Normoxic Hypoxic Subject Low Moderate Low Moderate Low Moderate Low Moderate 1 53.1 77.8 58.0 116.4 48.5 79.5 57.1 96.4 2 39.5 64.4 48.3 41.8 69.4 45.1 3 39.2 70.1 47.2 96.7 41.4 66.7 49.6 90.1 4 37.2 60.7 44.8 83.0 37.4 69.2 46.1 89.7 5 42.1 72.0 46.0 89.2 40.9 75.6 49.5 95.0 6 23.8 48.5 28.3 59.3 23.8 44.6 23.0 50.6 7 40.8 77.9 46.4 90.0 39.7 78.3 48.5 100.3 8 47.3 103.1 57.7 113.5 45.8 85.9 56.6 114.0 9 52.7 87.5 62.4 104.9 53.4 80.0 63.7 100.9 10 38.0 59.0 49.3 86.0 41.2 67.2 50.2 92.6 Mean 41.4 72.1 48.8 93.2 41.4 71.6 48.9 92.2 Standard Deviation 8.5 15.6 9.4 17.4 7.8 11.4 10.7 17.3 Table 6.6: Submaximal minute ventilation values during exercise before and after SDIH. Values are expressed in litresmin"1 to Mean Minute Ventilation Pre-IH Mean Minute Ventilation Post-IH LDIH Normoxi c Hypoxic Normox c Hypoxic Subject Low Moderate Low Moderate Low Moderate Low Moderate 1 54.1 78.8 60.0 119.5 44.5 78.8 51.9 98.8 2 35.1 60.7 42.9 44.3 66.9 47.1 3 36.8 59.0 38.2 83.9 41.8 65.1 45.2 90.8 4 42.5 70.0 51.3 106.6 36.9 64.1 47.9 81.9 5 38.7 70.2 41.5 76.2 41.1 66.6 45.8 84.4 6 22.9 42.6 24.9 52.6 22.0 45.6 25.0 56.6 7 38.6 77.8 47.5 100.2 43.6 82.2 49.4 103.0 8 46.9 80.5 50.8 100.5 47.5 81.2 52.1 99.3 9 48.2 80.5 54.7 95.8 49.5 77.9 57.8 96.7 10 39.9 65.8 51.4 89.3 40.4 72.8 50.8 93.1 Mean 40.4 68.6 46.3 91.6 41.2 70.1 47.3 89.4 Standard Deviation 8.5 12.1 9.9 19.4 7.6 11.0 8.7 14.1 Table 6.7: Submaximal minute ventilation values during exercise before and after LDIH. litresmin"1 Values are expressed in SDIH Subject Pre- Post- Delta 1 83.9 89.0 5.1 2 90.9 92.6 1.7 3 85.0 87.3 2.2 4 85.0 85.6 0.6 5 81.5 86.5 5.0 6 86.2 90.6 4.4 7 86.2 87.7 1.5 8 84.7 87.5 2.8 9 82.4 86.3 3.9 10 83.1 82.4 -0.6 Mean 84.9 87.6 2.7 Standard Deviation 2.6 2.8 1.9 LDIH Subject Pre- Post- Delta 1 86.9 84.5 -2.4 2 90.2 90.2 0.0 3 84.3 86.0 1.7 4 80.4 84.8 4.4 5 86.4 87.7 1.3 6 86.8 86.9 0.1 7 87.8 86.6 -1.2 8 86.4 87.7 1.3 9 89.2 86.8 -2.4 10 82.8 83.6 0.8 Mean 86.1 86.5 0.4 Standard Deviation 2.9 1.9 2.0 Table 6.8: Submaximal saturation values during hypoxic exercise before and expressed in %Sa02 after intermittent hypoxia. Values are 0\ Pre 3bMA Delta Pre 3bMA Delta Pre 3bMA Delta Pre 3bMA Delta Subject Pre-SD H 1 50.0 42.7 7.3 51.6 38.8 12.8 66.7 61.5 5.2 78.2 62.9 15.3 2 39.2 34.6 4.6 40.5 28.7 11.9 62.5 57.9 4.6 72.7 56.5 16.1 3 39.4 30.1 9.4 39.8 32.5 7.2 72.5 54.2 18.3 69.6 58.4 11.2 4 37.0 30.8 6.2 40.0 32.2 7.8 64.8 48.0 16.8 60.3 47.3 13.0 5 42.2 34.9 7.3 41.1 37.8 3.3 65.4 51.6 13.8 77.2 67.7 9.5 6 23.0 22.9 0.1 24.7 23.6 1.0 46.3 41.8 4.4 52.6 45.4 7.2 7 43.1 35.3 7.8 39.9 33.8 6.1 78.5 64.4 14.0 81.1 67.0 14.1 8 37.1 36.6 0.6 50.8 42.9 8.0 91.8 78.0 13.9 111.7 94.2 17.5 9 53.9 40.7 13.3 53.7 44.9 8.9 85.2 77.1 8.1 90.2 79.2 11.0 10 33.4 33.5 -0.1 39.2 38.0 1.2 53.2 49.9 3.3 59.3 57.2 2.1 Mean 39.8 34.2 5.6 42.1 35.3 6.8 68.7 58.4 10.2 75.3 63.6 11.7 SD 8.6 5.6 4.4 8.4 6.4 4.0 13.9 12.0 5.7 17.1 14.6 4.6 Post-SDIH 1 52.3 38.4 13.9 47.8 38.8 9.1 78.8 61.6 17.2 83.1 68.0 15.1 2 42.0 34.3 7.7 42.0 40.0 2.0 70.9 60.6 10.3 3 39.6 34.4 5.2 40.3 38.5 1.8 62.4 51.2 11.2 69.9 59.0 10.9 4 35.5 31.0 4.5 37.8 36.5 1.3 67.7 51.8 15.8 71.6 55.2 16.4 5 42.9 33.9 8.9 42.3 36.0 6.3 79.7 66.4 13.3 77.5 68.4 9.1 6 22.6 20.2 2.4 22.6 20.2 2.4 44.2 32.9 11.3 46.4 41.2 5.2 7 41.3 36.7 4.6 38.1 23.8 14.4 79.0 60.4 18.6 83.0 61.1 21.9 8 42.9 36.2 6.7 46.5 43.0 3.5 79.5 70.2 9.3 87.5 80.0 7.5 9 62.6 45.5 17.2 49.3 42.0 7.3 80.0 62.5 17.5 78.1 72.5 5.6 10 40.9 36.7 4.2 37.4 37.2 0.2 69.4 58.1 11.3 67.7 57.3 10.4 Mean 42.3 34.7 7.5 40.4 35.6 4.8 71.2 57.6 13.6 73.9 62.5 11.3 SD 10.3 6.4 4.7 7.6 7.6 4.4 11.3 10.4 3.4 12.2 11.3 5.5 Table 6.9: Hyperoxic Test ventilation values during normoxic exercise before and after SDIH. 3bMA = Three-breath moving average. Pre = mean ventilation for the 30 seconds prior to the challenge. Values are expressed in litresmin"1 Pre 3bMA Delta Pre 3bMA Delta Pre 3bMA Delta Pre 3bMA Delta Subject Pre-LD IH 1 55.3 40.3 15.0 52.0 48.0 4.0 74.7 58.1 16.7 83.5 61.7 21.8 2 36.2 26.7 9.5 31.8 28.0 3.8 63.3 52.6 10.7 60.1 53.9 6.3 3 33.2 33.3 0.0 39.0 32.7 6.3 59.4 50.8 8.6 62.5 50.3 12.2 4 41.1 29.1 12.0 44.1 30.4 13.6 68.6 62.6 6.0 75.2 63.4 11.8 5 40.3 35.2 5.1 37.0 35.3 1.7 70.8 53.0 17.8 75.7 55.5 20.3 6 22.6 20.6 2.1 21.5 18.1 3.4 42.9 39.4 3.4 42.1 37.9 4.3 7 37.5 31.5 6.0 40.5 32.1 8.4 68.9 64.5 4.4 84.6 64.8 19.9 8 49.1 38.2 10.9 48.5 42.5 6.0 76.8 68.6 8.2 82.8 73.8 9.0 9 48.0 36.0 12.0 47.1 39.1 8.0 79.7 68.0 11.7 85.2 73.0 12.2 10 41.3 34.0 7.4 41.0 36.0 5.0 65.9 62.1 3.7 66.7 62.3 4.5 Mean 40.5 32.5 8.0 40.3 34.2 6.0 67.1 58.0 9.1 71.9 59.6 12.2 SD 9.1 5.8 4.8 8.8 8.2 3.4 10.5 9.1 5.1 14.0 10.8 6.5 Post-LDIH 1 50.4 36.9 13.4 44.7 37.3 7.4 79.4 59.6 19.8 80.1 58.1 21.9 2 43.4 37.7 5.7 41.6 30.4 11.2 65.3 56.9 8.4 70.5 51.1 19.5 3 40.7 35.5 5.2 42.0 40.4 1.6 59.3 56.0 3.3 67.5 60.6 7.0 4 33.4 27.8 5.5 36.0 33.4 2.6 62.4 46.8 15.6 65.2 55.1 10.1 5 42.0 37.7 4.3 39.5 36.0 3.5 74.5 63.0 11.5 73.1 63.8 9.3 6 22.0 17.5 4.5 21.2 18.7 2.5 42.7 38.9 3.8 45.8 42.0 3.8 7 43.0 33.0 10.0 45.0 34.8 10.1 72.1 64.3 7.8 83.8 74.7 9.2 8 52.1 39.8 12.3 47.4 40.5 6.9 76.8 67.5 9.3 83.8 60.8 23.0 9 53.0 35.0 18.0 49.9 44.2 5.7 74.0 66.7 7.3 79.0 73.7 5.3 10 39.6 28.8 10.8 40.9 39.4 1.5 73.2 58.8 14.4 74.7 53.2 21.5 Mean 41.9 33.0 9.0 40.8 35.5 5.3 68.0 57.8 10.1 72.3 59.3 13.1 SD 9.3 6.7 4.7 8.0 7.1 3.5 11.0 9.0 5.3 11.3 10.0 7.5 Table 6.10: Hyperoxic Test ventilation values during normoxic exercise before and after LDIH. 3bMA = Three-breath moving average. Pre = mean ventilation for the 30 seconds prior to the challenge. Values are expressed in litresmin -1 Pre 3bMA Delta Pre 3bMA Delta Pre 3bMA Delta Pre 3bMA Delta Subject Pre-SD H 1 55.2 59.3 4.1 55.6 59.5 3.9 81.8 91.8 10.0 84.6 92.1 7.5 2 38.7 56.5 17.8 39.6 55.7 16.1 61.6 76.2 14.6 61.0 80.0 19.0 3 37.9 43.4 5.5 39.7 43.8 4.1 66.5 83.2 16.7 71.6 82.8 11.2 4 34.7 45.3 10.7 37.0 44.8 7.8 57.6 70.5 12.9 60.1 76.0 15.9 5 40.6 53.8 13.2 44.7 54.9 10.2 70.3 80.5 10.2 75.2 91.1 16.0 6 24.0 32.1 8.1 23.8 31.8 8.0 47.1 55.2 8.1 48.0 60.5 12.5 7 39.3 47.7 8.4 40.9 48.9 8.0 73.2 87.4 14.3 78.8 91.3 12.5 8 49.3 59.1 9.8 51.8 65.2 13.4 99.8 116.7 16.9 108.9 122.6 13.6 9 50.5 53.6 3.1 52.4 61.3 8.9 80.3 97.5 17.2 94.4 124.0 29.6 10 39.2 43.4 4.2 40.2 47.3 7.1 60.2 63.6 3.4 63.1 72.2 9.1 Mean 40.9 49.4 8.5 42.6 51.3 8.8 69.8 82.3 12.4 74.6 89.2 14.7 SD 8.9 8.6 4.6 9.2 10.0 3.8 14.9 17.6 4.5 18.1 20.4 6.2 Post-SDIH 1 44.9 58.6 13.6 48.8 58.2 9.4 77.9 91.1 13.2 78.2 95.0 16.8 2 42.1 47.8 5.7 41.1 59.8 18.7 67.8 80.1 12.3 3 42.0 47.2 5.2 43.5 51.6 8.1 66.3 84.1 17.8 68.2 80.2 11.9 4 38.0 53.9 15.8 38.2 53.1 14.9 68.4 88.8 20.4 69.2 93.6 24.4 5 38.0 45.3 7.3 40.4 47.3 6.9 67.5 83.0 15.5 77.6 86.3 8.6 6 26.8 27.9 1.1 23.0 38.6 15.7 42.0 48.2 6.2 46.0 50.7 4.7 7 41.0 53.7 12.7 38.2 54.5 16.3 72.5 93.5 21.0 78.8 108.6 29.8 8 45.9 66.4 20.6 48.0 63.0 15.0 85.0 95.9 10.9 91.5 105.7 14.1 9 53.8 64.1 10.3 47.9 61.3 13.4 76.2 90.0 13.8 85.8 97.3 11.5 10 40.5 45.7 5.1 46.1 51.5 5.4 62.7 69.0 6.3 68.9 72.8 3.9 Mean 41.3 51.1 9.8 41.5 53.9 12.4 68.6 82.4 13.7 73.8 87.8 14.0 SD 6.9 11.1 5.9 7.7 7.3 4.5 11.4 14.3 5.1 13.1 18.0 8.6 Table 6.11: Hypercapnic Test ventilation values during normoxic exercise before and after SDIH. 3bMA = Three-breath moving average. Pre = mean ventilation for the 30 seconds prior to the challenge. Values are expressed in litresmin"1 Pre 3bMA Delta Pre 3bMA Delta Pre 3bMA Delta Pre 3bMA Delta Subject Pre-LDIH 1 57.2 66.1 8.9 52.0 64.3 12.3 76.2 97.7 21.5 81.0 93.7 12.7 2 35.8 43.6 7.8 36.6 44.0 7.4 58.0 61.8 3.8 61.5 68.1 6.6 3 34.6 46.0 11.4 40.4 51.8 11.3 56.2 66.6 10.4 57.9 72.6 14.7 4 44.0 49.9 5.9 40.7 51.1 10.4 68.7 96.3 27.5 67.5 93.9 26.3 5 39.6 45.4 5.8 37.7 42.3 4.6 64.0 72.3 8.3 70.4 82.8 12.4 6 24.7 34.8 10.1 22.9 27.1 4.2 41.0 50.9 9.9 44.6 58.1 13.5 7 39.1 41.6 2.5 37.1 55.9 18.9 75.6 88.5 12.9 82.2 97.8 15.7 8 44.7 51.5 6.8 45.0 59.3 14.2 79.7 86.7 7.0 82.7 94.9 12.2 9 47.6 58.2 10.6 50.1 62.8 12.7 75.4 82.1 6.7 81.7 103.3 21.6 10 35.5 39.7 4.3 41.9 47.4 5.5 63.5 65.5 2.0 67.0 72.0 5.0 Mean 40.3 47.7 7.4 40.4 50.6 10.2 65.8 76.8 11.0 69.6 83.7 14.1 SD 8.8 9.2 2.9 8.1 11.1 4.7 11.9 15.7 7.9 12.7 15.2 6.3 Post-LDIH 1 38.1 53.1 14.9 44.7 57.1 12.3 74.3 84.0 9.7 81.5 96.7 15.2 2 49.4 51.2 1.8 42.9 51.4 8.5 64.9 80.3 15.4 66.7 81.7 14.9 3 41.1 47.9 6.8 43.6 50.6 7.0 64.0 75.9 11.9 69.5 80.7 11.2 4 39.3 46.7 7.5 39.0 46.3 7.3 65.2 80.0 14.8 63.4 84.7 21.3 5 43.8 49.0 5.2 39.1 45.0 5.8 58.4 64.3 5.9 60.3 67.8 7.5 6 21.8 25.7 3.9 22.9 26.8 3.9 46.6 62.3 15.7 47.4 57.7 10.2 7 41.9 54.8 12.9 44.4 58.6 14.2 89.7 116.9 27.2 83.1 114.9 31.8 8 45.3 55.9 10.5 45.0 59.9 14.9 81.5 98.1 16.6 82.7 98.8 16.1 9 45.0 53.7 8.7 50.3 58.3 8.0 70.8 75.7 4.9 88.0 101.2 13.2 10 39.7 45.5 5.8 41.4 47.9 6.5 67.3 81.3 14.0 76.2 76.7 0.5 Mean 40.5 48.3 7.8 41.3 50.2 8.8 68.3 81.9 13.6 71.9 86.1 14.2 SD 7.4 8.7 4.0 7.2 9.9 3.7 11.9 15.9 6.3 12.7 17.0 8.3 Table 6.12: Hypercapnic Test ventilation values during normoxic exercise before and after LDIH. 3bMA = Three-breath moving average. Pre = mean ventilation for the 30 seconds prior to the challenge. Values are expressed in litresmin"1 Pre 3bMA Delta Pre 3bMA Delta Pre | 3bMA Delta Pre 3bMA | Delta Subject Pre-SDIH 1 57.5 38.0 19.5 54.9 37.9 17.0 101.7 55.1 46.6 104.1 68.3 35.9 2 47.7 29.4 18.2 47.4 30.7 16.7 50.9 37.0 13.9 3 44.6 30.5 14.1 48.8 35.5 13.2 104.1 72.9 31.2 107.7 77.2 30.5 4 43.5 24.3 19.2 45.7 26.7 18.9 78.7 50.9 27.9 89.5 55.4 34.1 5 44.7 37.0 7.7 46.6 38.6 8.0 82.5 59.8 22.7 89.8 70.3 19.6 6 28.9 16.8 12.1 28.1 22.2 6.0 57.6 46.4 11.2 60.3 43.4 16.9 7 47.0 32.4 14.6 42.9 35.3 7.6 90.6 65.8 24.8 89.4 66.5 22.9 8 55.5 41.9 13.7 59.0 44.0 15.0 86.5 86.5 0.0 123.8 98.6 25.2 9 58.4 33.4 25.0 62.1 36.6 25.5 105.5 70.8 34.6 106.9 73.3 33.6 10 47.0 25.9 21.1 53.8 22.9 30.9 82.4 48.3 34.1 92.3 61.2 31.1 Mean 47.5 31.0 16.5 48.9 33.0 15.9 84.0 59.4 24.7 96.0 68.2 27.7 SD 8.6 7.4 5.0 9.6 7.2 7.9 18.4 14.8 13.5 17.7 15.3 6.9 Post-SDIH 1 58.1 43.9 14.2 56.8 39.9 16.9 98.7 66.9 31.8 99.5 74.4 25.0 2 46.3 21.3 24.9 42.6 31.6 11.0 3 49.6 26.1 23.6 51.3 39.2 12.1 83.8 55.5 28.3 89.5 63.9 25.6 4 45.1 23.9 21.1 46.6 27.3 19.3 87.3 63.5 23.8 94.6 64.0 30.6 5 50.1 39.8 10.3 51.8 41.3 10.5 97.8 78.9 19.0 99.9 79.2 20.8 6 21.8 15.2 6.6 23.9 18.2 5.8 51.3 34.8 16.4 55.5 37.9 17.6 7 47.9 33.0 14.9 49.2 33.0 16.2 99.1 69.4 29.7 105.0 74.2 30.8 8 55.1 37.9 17.2 58.3 37.7 20.6 102.8 81.2 21.6 120.0 94.1 25.9 9 61.9 32.7 29.2 66.4 36.9 29.5 99.7 58.2 41.5 98.2 69.0 29.2 10 51.4 33.5 17.8 49.4 25.2 24.3 93.0 49.0 44.0 93.1 64.2 28.9 Mean 48.7 30.7 18.0 49.6 33.0 16.6 90.4 61.9 28.5 95.0 69.0 26.1 SD 10.8 9.0 6.9 11.2 7.5 7.1 15.9 14.6 9.5 17.2 15.1 4.5 Table 6.13: Hyperoxic Test ventilation values during hypoxic exercise before and after SDIH. 3bMA = Three-breath moving average. Pre = mean ventilation for the 30 seconds prior to the challenge. Values are expressed in litresmin"1 Pre 3bMA | Delta Pre 3bMA Delta Pre 3bMA Delta Pre 3bMA Delta Subject Pre-LDIH 1 57.5 38.7 18.8 59.5 47.5 12.0 97.3 76.2 21.2 128.3 106.5 21.8 2 43.8 27.7 16.1 42.2 29.7 12.6 73.1 46.4 26.7 72.4 48.2 24.2 3 37.9 26.8 11.2 38.8 31.1 7.7 90.7 55.5 35.2 90.0 65.7 24.3 4 49.0 25.0 24.0 57.0 31.6 25.4 103.4 64.6 38.8 109.5 76.6 32.9 5 40.8 37.0 3.8 42.1 36.3 5.8 74.5 52.9 21.5 78.5 64.6 13.9 6 23.6 14.1 9.5 26.6 20.4 6.2 55.4 36.5 19.0 54.0 40.5 13.5 7 44.8 37.7 7.2 51.6 33.7 17.8 90.5 79.3 11.3 105.0 81.5 23.5 8 50.1 33.5 16.5 50.5 41.1 9.5 92.7 67.4 25.4 107.4 81.0 26.5 9 55.9 37.2 18.8 57.4 33.6 23.7 92.8 64.1 28.7 99.7 80.7 19.0 10 50.1 32.8 17.4 51.4 41.0 10.4 89.4 64.8 24.7 90.1 51.8 38.3 Mean 45.4 31.0 14.3 47.7 34.6 13.1 86.0 60.8 25.2 93.5 69.7 23.8 SD 9.8 7.7 6.2 10.2 7.5 7.0 14.2 13.1 7.9 21.3 19.6 7.7 Post-LDIH 1 50.5 38.6 11.9 52.1 37.3 14.8 93.5 70.6 22.9 99.5 74.7 24.8 2 47.4 34.1 13.3 46.2 32.2 14.1 3 43.4 35.4 8.0 48.7 31.0 17.8 88.3 59.8 28.4 99.8 58.8 41.0 4 47.9 21.2 26.6 46.7 26.1 20.5 84.7 38.1 46.6 81.4 35.8 45.6 5 46.0 37.4 8.5 47.6 38.6 9.0 88.2 62.4 25.8 95.6 78.0 17.5 6 23.6 19.8 3.8 25.0 15.5 9.5 53.7 34.1 19.6 56.4 37.5 18.9 7 51.0 39.2 11.8 48.9 31.9 16.9 88.2 71.5 16.7 104.9 95.6 9.3 8 51.8 35.7 16.1 51.3 32.1 19.2 91.3 70.4 20.9 100.1 78.6 21.4 9 54.4 28.6 25.9 58.2 40.5 17.7 90.6 60.1 30.5 100.1 68.6 31.6 10 50.1 32.5 17.5 50.6 30.7 19.9 98.1 54.9 43.2 104.0 80.8 23.2 Mean 46.6 32.2 14.3 47.5 31.6 16.0 86.3 58.0 28.3 93.5 67.6 25.9 SD 8.7 6.9 7.4 8.6 7.1 4.1 12.8 13.7 10.4 15.5 20.1 11.6 Table 6.14: Hyperoxic Test ventilation values during hypoxic exercise before and after LDIH. 3bMA = Three-breath moving average. Pre = mean ventilation for the 30 seconds prior to the challenge. Values are expressed in litresmin"1 to Pre 3bMA Delta Pre 3bMA Delta Pre 3bMA Delta Pre 3bMA Delta Subject Pre-SDIH 1 59.7 66.3 6.6 60.1 66.1 6.1 128.3 135.0 6.6 131.3 145.8 14.4 2 47.1 74.3 27.2 51.0 73.7 22.7 78.9 97.6 18.7 3 46.5 57.8 11.3 48.9 64.1 15.3 83.4 103.5 20.1 91.6 108.5 16.9 4 42.3 54.8 12.5 47.8 68.2 20.4 79.3 94.3 15.0 84.5 97.6 13.1 5 44.4 52.3 7.9 48.3 53.7 5.4 86.5 105.1 18.6 98.1 101.0 3.0 6 28.7 37.6 8.9 27.5 41.6 14.1 57.9 61.9 4.0 61.6 68.8 7.2 7 49.3 55.2 5.8 46.3 54.8 8.5 84.7 97.7 13.0 95.2 101.1 5.8 8 58.3 79.3 21.0 57.9 80.1 22.2 123.3 135.7 12.4 120.1 135.3 15.2 9 66.1 83.3 17.2 63.0 71.2 8.2 99.4 121.0 21.6 107.7 134.8 27.1 10 45.7 51.9 6.2 50.7 58.7 7.9 78.9 97.3 18.4 90.6 98.1 7.6 Mean 48.8 61.3 12.5 50.1 63.2 13.1 90.1 104.9 14.8 97.9 110.1 12.3 SD 10.5 14.2 7.2 9.8 11.3 6.8 21.5 21.7 5.9 20.3 24.2 7.3 Post-S 3IH 1 55.0 67.6 12.6 58.5 76.6 18.2 94.0 118.6 24.6 93.4 109.6 16.2 2 44.8 64.7 19.9 46.8 56.6 9.8 3 46.6 58.7 12.2 50.8 60.1 9.3 92.0 114.8 22.7 95.1 130.2 35.1 4 44.6 75.1 30.5 48.2 71.2 22.9 85.5 110.3 24.8 91.2 106.6 15.4 5 47.4 54.8 7.4 48.7 53.7 5.0 85.4 104.3 18.9 96.7 103.0 6.2 6 21.9 32.6 10.7 24.6 30.5 5.9 46.7 63.9 17.3 48.7 51.8 3.1 7 47.0 60.2 13.3 49.9 55.4 5.5 91.8 110.6 18.9 105.3 124.1 18.8 8 55.5 84.0 28.5 57.2 78.4 21.2 113.6 131.4 17.8 119.5 140.2 20.6 9 60.8 75.3 14.6 65.6 72.5 6.9 100.7 110.7 10.0 105.0 124.1 19.1 10 51.4 55.8 4.4 48.7 57.0 8.3 92.2 95.6 3.4 92.3 105.9 13.6 Mean 47.5 62.9 15.4 49.9 61.2 11.3 89.1 106.7 17.6 94.1 110.6 16.5 SD 10.5 14.3 8.5 10.7 14.3 6.8 18.1 18.8 7.0 19.3 25.4 9.1 Table 6.15: Hypercapnic Test ventila ion values during hypoxic exercise before and after SDIH. 3bMA = Three-breath moving average. Pre = mean ventilation for the 30 seconds prior to the challenge. Values are expressed in litresmin"1 Pre 3bMA Delta Pre 3bMA Delta | Pre 3bMA Delta | Pre 3bMA Delta Subject Pre-LD H 1 60.1 85.5 25.4 62.9 74.1 11.2 113.0 131.5 18.5 139.4 164.4 24.9 2 42.9 55.0 12.2 42.8 48.8 6.1 65.8 83.1 17.2 70.3 83.8 13.5 3 34.4 51.6 17.2 41.6 52.2 10.6 73.0 98.3 25.2 81.8 102.7 20.9 4 47.6 60.9 13.3 51.7 69.5 17.8 102.1 120.4 18.3 111.3 122.1 10.8 5 40.6 44.2 3.6 42.6 51.1 8.5 68.8 99.3 30.5 83.1 89.5 6.4 6 24.3 38.8 14.5 25.0 29.1 4.1 49.2 66.2 16.9 51.5 69.8 18.3 7 48.1 66.9 18.7 45.5 61.1 15.6 98.8 113.4 14.7 106.4 128.3 21.9 8 51.6 69.8 18.2 51.1 71.2 20.1 98.8 112.7 13.9 103.0 125.0 22.0 9 52.8 59.2 6.4 52.5 63.7 11.2 91.0 107.2 16.2 99.7 110.6 11.0 10 53.9 57.1 3.2 50.0 64.3 14.3 85.4 94.9 9.5 92.1 107.9 15.8 Mean 45.6 58.9 13.3 46.6 58.5 11.9 84.6 102.7 18.1 93.9 110.4 16.5 SD 10.5 13.3 7.2 9.9 13.5 5.1 19.8 18.8 5.9 24.2 26.8 6.0 Post-LDIH 1 51.8 56.8 5.0 53.2 58.8 5.6 92.6 110.7 18.1 109.5 122.0 12.5 2 45.1 46.7 1.6 49.7 66.3 16.6 3 42.9 65.8 22.9 45.8 66.9 21.1 81.5 97.3 15.8 93.6 111.2 17.7 4 47.5 58.8 11.3 49.5 58.1 8.6 78.1 100.6 22.5 83.6 96.9 13.4 5 43.5 53.0 9.5 46.1 51.4 5.3 73.8 89.4 15.6 80.1 90.4 10.3 6 25.0 33.5 8.6 26.3 32.9 6.6 58.4 63.3 4.9 57.8 68.6 10.8 7 52.0 61.7 9.7 45.6 66.9 21.3 112.6 126.0 13.5 106.3 144.8 38.5 8 52.1 73.8 21.7 53.2 70.8 17.6 97.8 111.8 13.9 108.1 122.6 14.5 9 57.0 63.1 6.0 61.7 69.2 7.5 98.4 110.4 12.1 97.6 113.3 15.7 10 51.8 68.8 17.0 50.7 56.9 6.2 81.5 94.2 12.7 89.0 101.7 12.7 Mean 46.9 58.2 11.3 48.2 59.8 11.7 86.1 100.4 14.3 91.7 107.9 16.2 SD 8.9 11.6 7.1 9.0 11.3 6.7 16.0 17.8 4.8 16.6 21.9 8.7 Table 6.16: Hypercapnic Test ventilation values during hypoxic exercise before and after LDIH. 3bMA = Three-breath moving average. Pre = mean ventilation for the 30 seconds prior to the challenge. Values are expressed in litresmin"1 Subject SDIH Protocol Day 1 SDIH Protocol Day 8 Ramp Time (s) Peak V02 Peak Ve Ramp Time (s) Peak V02 Peak Ve 1 9 3.06 122.3 64 2.92 131.9 2 0 0 3 0 106.5 150 103.3 4 134 83.3 62 96.1 5 205 2.87 103.3 166 2.84 94.4 6 220 84.0 217 95.8 7 50 174 8 130 2.43 141.4 165 2.65 166.6 9 210 3.81 131.8 330 4.30 174.7 10 160 3.23 111.7 154 3.28 117.8 Mean 111.8 3.1 110.5 148.2 3.2 122.6 Standard Deviation 89.8 0.5 20.9 91.6 0.7 32.4 „W I I _ _ . w. w. « w and after SDIH. Ventilation and oxygen consumption values are expressed in litresmin"1 Subject LDIH Protocol Day 1 LDIH Protocol Day ( i Ramp Time (s) Peak V02 Peak Ve Ramp Time (S) Peak V02 Peak Ve 1 60 3.26 132.6 142 3.07 97.2 2 30 0 N/A 3 142 111.9 150 102.3 4 190 136.6 143 89.9 5 216 2.60 97.3 232 2.61 110.5 6 188 89.2 176 88.5 7 148 155 8 180 2.86 149.3 225 3.60 154.8 9 244 3.44 116.2 310 3.35 122.0 10 217 3.24 118.3 241 3.29 125.1 Mean 161.5 3.1 118.9 177.4 3.2 111.3 Standard Deviation 69.0 0.3 20.1 83.0 0.4 22.2 Table 6.18: Peak Ramp time, oxygen consumption V02, and ventilation during a graded exercise test in hypoxia before and after LDIH. Ventilation and oxygen consumption values are expressed in litresmin"1 <3\ 

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