Cardiovascular and cerebrovascular responses ... - Wiley Online Library

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J Physiol 587.13 (2009) pp 3287–3299

Cardiovascular and cerebrovascular responses to acute hypoxia following exposure to intermittent hypoxia in healthy humans Glen E. Foster1,5 , Julien V. Brugniaux1,5 , Vincent Pialoux1,5 , Cailean T. C. Duggan1,5 , Patrick J. Hanly2,5 , Sofia B. Ahmed2,6 and Marc J. Poulin1,3,4,5,6 Departments of 1 Physiology & Pharmacology, 2 Medicine and 3 Clinical Neurosciences, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada 4 Faculty of Kinesiology, University of Calgary, Calgary, Alberta, Canada 5 Hotchkiss Brain Institute, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada 6 Libin Cardiovascular Institute of Alberta, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada

Intermittent hypoxia (IH) is thought to be responsible for many of the long-term cardiovascular consequences associated with obstructive sleep apnoea (OSA). Experimental human models of IH can aid in investigating the pathophysiology of these cardiovascular complications. The purpose of this study was to determine the effects of IH on the cardiovascular and cerebrovascular response to acute hypoxia and hypercapnia in an experimental human model that simulates the hypoxaemia experienced by OSA patients. We exposed 10 healthy, male subjects to IH for 4 consecutive days. The IH profile involved 2 min of hypoxia (nadir P ET,O2 = 45.0 mmHg) alternating with 2 min of normoxia (peak P ET,O2 = 88.0 mmHg) for 6 h. The cerebral blood flow response and the pressor responses to hypoxia and hypercapnia were assessed after 2 days of sham exposure, after each day of IH, and 4 days following the discontinuation of IH. Nitric oxide derivatives were measured at baseline and following the last exposure to IH. After 4 days of IH, mean arterial pressure increased by 4 mmHg (P < 0.01), nitric oxide derivatives were reduced by 55% (P < 0.05), the pressor response to acute hypoxia increased (P < 0.01), and the cerebral vascular resistance response to hypoxia increased (P < 0.01). IH alters blood pressure and cerebrovascular regulation, which is likely to contribute to the pathogenesis of cardiovascular and cerebrovascular disease in patients with OSA. (Received 26 February 2009; accepted after revision 30 April 2009; first published online 5 May 2009) Corresponding author M. J. Poulin: Department of Physiology and Pharmacology, Faculty of Medicine, University of Calgary, HMRB-210, 3330 Hospital Drive NW, Calgary, AB, T2N 4N1, Canada. Email: [email protected] Abbreviations AHCR, acute hypercapnic response; AHR, acute hypoxic response; CVR, cerebrovascular resistance; DBP, diastolic blood pressure; HRT, heart rate; IH, intermittent hypoxia; MAP, mean arterial pressure; ODI, oxygen desaturation index; OSA, obstructive sleep apnoea; P ET,O2 , end-tidal partial pressure of oxygen; P ET,CO2 , end-tidal partial pressure of carbon dioxide; S a,O2 , arterial oxyhaemoglobin saturation; SBP, systolic blood pressure.

Obstructive sleep apnoea (OSA) is a chronic medical disorder during which patients experience intermittent hypoxia (IH), often for many years prior to diagnosis and treatment (Somers et al. 2008). The exposure to chronic IH is thought to be responsible for many of the long-term cardiovascular consequences associated with OSA (Somers et al. 2008), including systemic hypertension (Morrell et al. 2000; Nieto et al. 2000; Peppard et al. 2000), myocardial infarction (Hung et al. 1990; Shahar et al. 2001), and stroke (Arzt et al. 2005; Yaggi et al. 2005; Sahlin et al. 2008). Although epidemiological studies have highlighted these associations, our understanding of the C 2009 The Authors. Journal compilation C 2009 The Physiological Society

underlying mechanisms is incomplete. A variety of animal and human models of IH have been developed to evaluate potential mechanisms for the association between IH and cardiovascular disease (Foster et al. 2007b). However, few, if any, of them replicate the pattern of IH experienced by patients with OSA. OSA patients have repeated apnoeas throughout the night that are associated with arterial oxyhaemoglobin desaturation between 5 and 10% (Somers et al. 2008). Although the diagnosis of sleep apnoea is based on an apnoea hypopnoea index of 5 or more per hour of sleep, the risk of cardiovascular complications increases DOI: 10.1113/jphysiol.2009.171553

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as apnoea frequency rises (Somers et al. 2008). Previous studies of patients with OSA suggest that IH increases resting blood pressure (Hla et al. 1994; Young et al. 1997; Narkiewicz et al. 1998b), sympathetic nervous system activity (Somers et al. 1995; Narkiewicz et al. 1998a,b), and circulating components such as angiotensin-II (Moller et al. 2003) and endothelin-1 (Phillips et al. 1999). A reduced cerebrovascular response to hypoxia (Foster et al. 2007a) and reduced plasma levels of nitric oxide derivatives (Ip et al. 2000; Schulz et al. 2000) have also been reported in patients with OSA. Nitric oxide is an important vasodilator in the cerebral circulation and its bioavailability is reduced by the formation of reactive oxygen species (Cohen, 1995; Lavie, 2003). If IH is responsible for such alterations in vascular function, it would be expected to alter resting blood pressure, the pressor response to hypoxia and the cerebrovascular response to hypoxia. Reductions in nitric oxide bioavailability may be one mechanism contributing to altered vascular function. All of these phenomena that have been described in OSA patients are likely to contribute to the development of cardiovascular and cerebrovascular disease. However, interpretation of data obtained from studies on patients with OSA are often confounded by coexisting chronic cardiovascular disease and acute physiological changes that accompany OSA such as sleep fragmentation and alteration of intra-thoracic pressure. Consequently, it is difficult to identify underlying pathophysiological mechanisms that are responsible for specific clinical outcomes. The development of a healthy human model of IH provides the opportunity to assess the direct effects of IH on cardiovascular and cerebrovascular control in the absence of coexisting disease, sleep fragmentation, hypercapnia, and intra-thoracic pressure fluctuations. The purpose of this study was to determine the effects of IH on the cardiovascular and cerebrovascular responses to acute hypoxia and hypercapnia in an experimental human model that replicates the type of hypoxia that is experienced by patients with OSA. In addition, the concentration of nitric oxide derivatives in the blood was measured to determine the extent to which IH affects nitric oxide bioavailability. We hypothesized that IH increases resting blood pressure and the pressor response to acute hypoxia, and decreases the reduction in cerebrovascular resistance (CVR) in response to acute hypoxia, and that these changes may be mediated in part by a reduction in nitric oxide bioavailability. Methods Ethical approval

All experimental procedures and protocols were approved by the Conjoint Health Research Ethics Board at the

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University of Calgary and conformed to the Declaration of Helsinki. All subjects provided written, informed consent prior to their participation. Subjects

We studied 10 healthy, male human volunteers between 21 and 36 years of age. All subjects were acclimatized to an altitude of 1100 m, which corresponds to the elevation of the research laboratory (Calgary, Alberta). Subjects were excluded from participation if they had a history of pulmonary, cardiovascular, or neurological disease, sleep apnoea, diabetes, or smoking, or were hypertensive (systolic > 140 mmHg; diastolic > 90 mmHg). These criteria helped to ensure that all subjects studied were healthy and did not have any medical conditions that could confound our outcome measurements. Experimental protocol

The experimental protocol is displayed in Fig. 1. Subjects were required to attend the cerebrovascular physiology laboratory on seven different days over a 13 day period. On all experimental days the subjects attended the laboratory at approximately 07.30 h to prepare for the day of testing. On days minus four (Day –4) and zero (Day 0), subjects were exposed to intermittent normoxia (i.e. sham IH) for 6 h in a purpose-built chamber. After the exposure, a venous blood sample was taken. Approximately 30 min following the exposure to intermittent normoxia an acute hypoxia and hypercapnia test was conducted to assess the cardiovascular and cerebrovascular responses to each stimulus. On Days 1–4, subjects were exposed to 6 h of intermittent hypoxia. A venous blood sample was taken on Day 4 following exposure to IH. The cardiovascular and cerebrovascular responses to acute hypoxia and hypercapnia were also assessed after exposure to IH on Days 1–4. Subjects returned to the laboratory 4 days after their last exposure to IH on Day 8, where a venous blood sample was collected and the physiological responses to acute hypoxia and hypercapnia were assessed for the last time. The same investigators conducted all experimental procedures, and the exposures to IH and to acute hypoxia and hypercapnia occurred at the same time of day. All subjects completed daily dietary and physical activity diaries to ensure that their diet and exercise schedule remained consistent throughout the protocol. In addition, 8 of 10 subjects were monitored by an actigraphy system (Actiwatch, Respironics Inc., Bend, OR, USA) throughout the protocol providing a second index of daily activity and, also, a measure of their sleep/wake schedule. C 2009 The Authors. Journal compilation C 2009 The Physiological Society

Intermittent hypoxia and vascular control

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Experimental techniques Baseline measurements. Resting measurements of the end-tidal partial pressures of O 2 (P ET,O2 ) and CO 2 (P ET,CO2 ), blood pressure, heart rate and cerebral blood flow were collected while the subject rested quietly and comfortably in a semi-seated position. Although resting air-breathing data were collected for a 10 min period, only the last 5 min was used in the analysis of baseline values. Respired gases were sampled via a fine catheter at the mouth at a rate of 20 ml min−1 and analysed for P O2 and P CO2 by mass spectrometry (AMIS 2000, Innovision, Odense, Denmark). A computer was used to acquire values for P O2 and P CO2 every 10 ms and the values for P ET,O2 and P ET,CO2 were determined and recorded for each breath using dedicated software (Chamber V2.43, University Laboratory of Physiology, Oxford, UK). Blood pressure was recorded at the finger by photoplethysmography (Portapress, TPD Biomedical Instrumentation, Amsterdam, Netherlands) and was calibrated against measurements taken from an automated arm cuff. Resting normoxic blood pressure was taken as the mean of three values taken from the automated blood pressure monitoring device (Dinamap; Johnson and Johnson Medical, Inc., New Brunswick, NJ, USA) provided that the three values were within

5% of each other. If values were not within 5% an extra measurement was taken. Heart rate (HRT) was measured by three-lead electrocardiogram (Micromon, 7142B monitor, Kontron Medical, UK), and arterial oxyhaemoglobin saturation (S a,O2 ) by pulse oximetry (3900 Datex-Ohmeda, Louisville, CO, USA). Several indices of cerebral blood flow velocity were measured in the middle cerebral artery by transcranial Doppler ultrasound (TC22, SciMed, Bristol, UK) and all were averaged over the cardiac cycle. These indices of cerebral blood flow include the velocity associated with the maximum frequency of the Doppler shift (V¯ P ) (proportional to axial flow velocity if flow is laminar and vessel diameter is constant), the velocity associated with the intensity-weighted mean frequency of the Doppler spectrum (V¯ IWM ) (proportional to overall blood flow provided that vessel diameter is constant and is independent of the complexity of the flow pattern), the total power of the Doppler spectrum (P¯ ; arbitrary units) (proportional to the cross sectional area of the vessel) and the flow index (P¯ V¯ IWM ; arbitrary units) (product of V¯ IWM and P¯ ; a surrogate measure for overall blood flow). All cardiovascular parameters were acquired every 10 ms and the values for each determined beat-by-beat by specifically designed computer software (Chamber V2.43).

Figure 1. Experimental protocol Displays the daily experimental protocol and the experimental timeline. IH, intermittent hypoxia. C 2009 The Authors. Journal compilation C 2009 The Physiological Society

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Measurement of the cardiovascular and cerebrovascular response to acute hypoxia and hypercapnia. The

protocol began with a 7.5 min lead-in period during which the subject breathed through a mouthpiece with the nose occluded by a nose clip. During the lead-in period P ET,O2 was maintained at 88.0 mmHg and P ET,CO2 was held at 1.5 mmHg above the normoxic P ET,CO2 . After the lead-in period the level of P ET,O2 was decreased in six 90 s steps (75.2, 64.0, 57.0, 52.0, 48.2, 45.0 mmHg). These levels were calculated to provide an equal decrement in S a,O2 by using the relationship between P ET,O2 and S a,O2 described by Severinghaus (1979). After the last 90 s step in P ET,O2 the level of P ET,O2 was returned to 88.0 mmHg for 5 min after which the level of P ET,CO2 was increased to +9 mmHg above resting normoxic P ET,CO2 . This level of hypercapnia was maintained for 5 min before the test ended. Accurate control of the end-tidal gases was achieved using the technique of dynamic end-tidal forcing (BreatheM V2.40, University Laboratory of Physiology, Oxford, UK) as previously described (Ainslie & Poulin, 2004). Throughout acute hypoxia and hypercapnia testing the following parameters were recorded: systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP), HRT, V¯ P , V¯ IWM , P¯ and P¯ V¯ IWM . In addition, cerebrovascular resistance (CVR) was calculated as MAP indexed against V¯ P (MAP/V¯ P ; mmHg (cm s−1 )−1 ). All cardiovascular parameters were acquired every 10 ms and the values for each determined beat-by-beat by specifically designed software (BreatheM V2.40, University Laboratory of Physiology, Oxford, UK). The acute hypoxic cerebral blood flow response (AHRV¯ P , AHRV¯ IWM , AHRP¯ V¯ IWM , and AHR CVR ) and blood pressure response (AHR SBP , AHR DBP , AHR MAP ) were measured as the slope of the linear regression between the percent change in each variable and S a,O2 . A single regression was determined based on the data averaged over the last 30s of each step in P ET,O2 for all subjects. The acute hypercapnic cerebral blood flow response (AHCRV¯ P , AHCRV¯ IWM , AHCRP¯ V¯ IWM , AHCR CVR ) and blood pressure response (AHCR SBP , AHCR DBP , AHCR MBP ) were determined as the percentage change in each parameter indexed over the change in P ET,CO2 . Exposure to sham IH and IH. Subjects were exposed to 6 h of continuous cycles of 2 min of hypoxia (nadir P ET,O2 = 45.0 mmHg) and 2 min of normoxia (peak P ET,O2 = 88.0 mmHg). P ET,CO2 was not controlled during episodes of IH. To create the IH paradigm subjects sat in a normobaric chamber. In this chamber, the gas composition can be altered by adding either nitrogen or oxygen, and by adding or removing carbon dioxide. On days of exposure to IH, the gas composition within the chamber was set at a level resulting in a P ET,O2 of 45.0 mmHg. Periods of normoxia were elicited by

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delivering 100% oxygen to the subject through an oxygen diffuser (Oxymask, Southmedic, Barrie, Ontario, Canada) worn at the mouth. The oxygen flow rate was set such that a P ET,O2 of 88.0 mmHg resulted. When oxygen flow through the diffuser was zero the subject breathed the gas composition of the chamber, which corresponded to a P ET,O2 of 45.0 mmHg. A computer controlled gas solenoid valve was used to turn the flow of oxygen through the diffuser on and off at 2 min intervals. Two-minute bouts of hypoxia were chosen because this was the time necessary for subjects to fully equilibrate with the gas composition of the chamber and elicited the largest drop in S a,O2 in a safe manner. During episodes of IH, respired gas was sampled from a nasal cannula and analysed by a dual oxygen and carbon dioxide analyser (NormocapOxy, Datex-Ohmeda) for P O2 and P CO2 . These values were acquired every 10 ms by a computer, and P ET,O2 and P ET,CO2 were identified and recorded for each breath using a computer and dedicated software (Chamber V2.43). S a,O2 was recorded continuously throughout the exposure at the ear by pulse oximetry (3900 Datex-Ohmeda). On Day 1 and Day 4 during the first and last 30 min of the exposure, blood pressure (SBP, DBP, MAP), HRT and cerebral blood flow indices (V¯ P , V¯ IWM , P¯ , P¯ V¯ IWM , CVR) were collected continuously. Data from each cycle of normoxia to hypoxia were interpolated at a 1 s interval, overlaid, and averaged to create a single 4 min cycle of normoxia and hypoxia using software designed in-house created in Matlab (V7.4.0.287, The MathWorks, Inc., Natick, MA, USA). Four 15 s averages were then determined at 30 s, 60 s, 90 s and 120 s throughout the cycle. The identical experimental set-up was used during sham IH exposure (intermittent normoxia), except the chamber was not hypoxic and room air was delivered through the oxygen diffuser rather than 100% oxygen. The oxygen desaturation index (ODI) was calculated as the number of times the level of S a,O2 was reduced by 4% or more per hour. The ODI is similar to the apnoea–hypopnoea index, which is conventionally used to diagnose OSA.

Venous blood sample measurements. On Day –4, Day 0, Day 4, and Day 8 a venous puncture was made in the antecubital vein immediately prior to acute hypoxia and hypercapnia testing, and 10 ml of blood was collected in serum separator tubes. The blood was centrifuged and the serum was separated into 0.5 ml aliquots and frozen at −80◦ C for subsequent analysis. The derivatives of nitric oxide breakdown, nitrites and nitrates, were measured in the serum using a commercially available kit (Cayman Chemical Company, Ann Arbor, MI, USA) based on methods previously described (Green et al. 1982). The sum of nitrite and nitrate in the plasma (NOx) is considered an index of nitric oxide production (Ohta et al. 2005). C 2009 The Authors. Journal compilation C 2009 The Physiological Society


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Table 1. Resting ventilatory, cardiovascular, and cerebrovascular parameters during air-breathing and isocapnic euoxia

Resting air-breathing data P ET,O2 (mmHg) P ET,CO2 (mmHg) V¯ P (cm s−1 ) V¯ IWM (cm s−1 ) P¯ (arbitrary units) P¯ V¯ IWM (arbitrary units) NOx (μmol l−1 )

Baseline

Day 1

Day 2

Day 3

Day 4

Day 8

86.5 ± 0.7 36.4 ± 0.5 51.0 ± 3.2 32.3 ± 2.7 3.02 ± 0.10 97.9 ± 10.2 19.9 ± 2.9

88.0 ± 0.8 35.1 ± 0.5∗ 51.5 ± 3.3 33.2 ± 2.5 3.05 ± 0.13 101.9 ± 9.6

89.1 ± 1.3 34.0 ± 0.6∗ 52.0 ± 3.0 34.2 ± 2.3 2.77 ± 0.19 95.0 ± 10.8

89.0 ± 1.2 34.1 ± 0.7 51.0 ± 3.3 32.7 ± 2.6 3.18 ± 0.09 105.1 ± 10.4

88.3 ± 0.7 34.3 ± 0.6∗ 50.1 ± 3.3 31.5 ± 2.6 3.07 ± 0.15 96.9 ± 9.7 8.9 ± 2.0∗

87.0 ± 1.2 35.9 ± 0.6 49.7 ± 4.0 32.2 ± 2.9 3.26 ± 0.14 104.6 ± 10.8 25.6 ± 5.9

88.0 ± 0.3 36.4 ± 0.4† 96.6 ± 0.3 57.1 ± 2.3 51.9 ± 3.5 33.2 ± 2.8 3.10 ± 0.12 103.7 ± 10.7 1.63 ± 0.12

87.4 ± 0.3 35.8 ± 0.5∗ 96.7 ± 0.3 58.0 ± 2.4 52.8 ± 2.7 34.8 ± 2.2 2.76 ± 0.18 97.1 ± 11.2 1.59 ± 0.13

87.6 ± 0.6 35.6 ± 0.5∗ 97.1 ± 0.3 56.8 ± 2.0 52.2 ± 3.3 33.2 ± 2.7 3.25 ± 0.12 108.7 ± 10.8 1.56 ± 0.11

87.8 ± 0.3 35.6 ± 0.6∗ 97.2 ± 0.4 57.3 ± 2.6 52.4 ± 2.7 32.7 ± 2.4 3.06 ± 0.24 100.9 ± 11.3 1.66 ± 0.08

87.4 ± 0.6 37.2 ± 0.4 96.3 ± 0.3 56.9 ± 3.4 50.9 ± 3.7 33.0 ± 2.8 3.33 ± 0.12 108.9 ± 9.8 1.58 ± 0.14

Resting isocapnic euoxia data 87.5 ± 0.3 P ET,O2 (mmHg) 37.8 ± 0.4 P ET,CO2 (mmHg) 96.8 ± 0.3 S a,O2 (%) HRT (beats min−1 ) 54.3 ± 3.4 V¯ P (cm s−1 ) 52.0 ± 2.8 32.9 ± 2.5 V¯ IWM (cm s−1 ) P¯ (arbitrary units) 3.06 ± 0.12 P¯ V¯ IWM (arbitrary units) 101.3 ± 10.1 CVR (mmHg cm−1 s−1 ) 1.71 ± 0.14 ∗P

< 0.05, †P < 0.01 compared to baseline. Definition of abbreviations: P ET,O2 , end-tidal partial pressure of oxygen; P ET,CO2 , end-tidal partial pressure of carbon dioxide; V¯ P , mean peak cerebral blood flow velocity; V¯ IWM , cerebral blood flow velocity intensity weighted mean; P¯ , mean power index; P¯ V¯ IWM , cerebral blood flow index; NOx, sum of nitrite and nitrate; S a,O2 , arterial oxyhaemoglobin saturation; HRT, heart rate; CVR, cerebrovascular resistance.

Statistical analysis

All data are expressed as means ± S.E.M. Data were compared using repeated-measures analysis of variance (SPSS, v. 16.0; SPSS Inc., Chicago, IL, USA). Since data on Day –4 and Day 0 were not statistically different a combined baseline was created for all parameters. When significant F-ratios were detected, Sidak’s post hoc test was applied to determine where the differences were in comparison to baseline. Linear regressions from each day were compared to a combined baseline and multiple partial F-tests (tests of coincidence and parallelism) were performed to assess differences in each relationship (Klienbaum et al. 1998), with two-tailed significance levels of 0.05.

Resting and isocapnic cardiovascular and cerebrovascular parameters

¯ P , V¯ IWM , P¯ and Measurements at rest. P ET,O2 , P ETCO2 , V

P¯ V¯ IWM were measured at rest during air breathing (i.e. normoxia) prior to acute testing (Table 1). There was a significant effect of time for P ET,CO2 (P < 0.001). Post hoc analysis revealed a significant reduction in P ET,CO2 on Days 1, 2, and 4. P ET,O2 and all indices of cerebral blood flow were unchanged by exposure to IH. The sum of nitric oxide by-products (NOx) in the serum was significantly reduced by exposure to IH (P < 0.05) and returned to baseline 4 days after the end of IH (P = 0.61). Resting systolic, diastolic, and mean arterial pressures were assessed prior to acute testing (Fig. 2). Systolic blood pressure tended to increase following 4 days of IH (P = 0.056). Both diastolic (P = 0.001) and mean arterial pressures (P = 0.002) were elevated after exposure to IH and returned to baseline by Day 8 (recovery).

Results Subjects

Ten subjects (29.4 ± 1.6 years, BMI 25.2 ± 0.4 kg m−2 ) completed each day of the experimental protocol. Subjects maintained a steady diet, activity level and sleep schedule throughout the experimental protocol (i.e. n = 13 days). Actigraphy revealed no significant differences in mean daily activity scores (P = 0.90) or in sleep/wake schedule (movement and fragmentation index; P = 0.81). C 2009 The Authors. Journal compilation C 2009 The Physiological Society

Measurements during isocapnic euoxia. Table 1 displays the resting cardiovascular and cerebrovascular parameters during isocapnic euoxia, the lead-in period to the assessment of the responses to acute hypoxia and hypercapnia. P ET,CO2 was reduced after 1 day of IH and remained at this level throughout the remaining days of IH (P < 0.05). No other respiratory, cardiovascular, and cerebrovascular parameter was changed by exposure to IH.

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Intermittent hypoxia exposure

The significant reduction in P ET,CO2 observed prior to acute testing was sustained overnight, as significant reductions in P ET,CO2 (P = 0.006) were observed between that measured in the morning prior to the Day 1 exposure (34.1 ± 0.7 mmHg) and that measured before the Day 4 exposure (32.4 ± 0.5 mmHg). Significant reductions in

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V¯ P (P = 0.02) were also observed pre-exposure on Days 1 and 4 (55.0 ± 3.5 and 51.9 ± 3.7 cm s−1 , respectively). A representative sample of the IH exposure is displayed in Fig. 3. This figure shows the fluctuations in P ET,O2 , S a,O2 and P ET,CO2 which occur throughout an entire 6 h protocol in one subject, and the resulting fluctuations in MAP and V¯ P which were measured during the first and last 30 min of exposure. IH elicited significant reductions in S a,O2 such that across all days of the protocol the ODI was 14.8 ± 0.1 events per hour, the mean minimum S a,O2 was 83.3 ± 0.5%, and the percentage of time spent below 90% and 85% S a,O2 was 38.7 ± 1.7% and 10.1 ± 2.4% of the study duration, respectively. The mean maximum S a,O2 was 95.9 ± 0.1%, which demonstrates that all subjects adequately re-oxygenated after each bout of hypoxia. Figure 4 displays the P ET,O2 , P ET,CO2 , S a,O2 , V¯ P , MAP and CVR during the first and last six cycles on Day 1 and Day 4 of IH. The P ET,O2 and S a,O2 were similar in the mornings and afternoons on Day 1 and Day 4. P ET,CO2 was significantly decreased on Day 4 compared to Day 1 (P < 0.01). There was a trend for a day effect for V¯ P , such that V¯ P was higher on day 4 (P = 0.053). This finding is driven by a larger increase in V¯ P in the afternoon of Day 4. A significant time of day effect was found for both MAP and CVR (P < 0.05) such that both were higher in the afternoon on Day 1 and Day 4 compared to the morning. Note that the intermittent hypoxia exposure was poikilocapnic and the increases in CVR when S a,O2 decreases are likely to reflect the influence of concomitant decreases in end-tidal P CO2 . Acute hypoxic response

Figure 5 displays the linear relationships between SBP, DBP, MAP and CVR and S a,O2 for the combined baseline and Day 4. Exposure to IH significantly increased the slope of the regressions relating SBP, DBP and MAP to S a,O2 . This finding is true for all days of IH exposure. The slope of the regression relating CVR and S a,O2 was significantly increased. Regression slopes tended to normalize for all variables on Day 8 testing. There were no changes in P¯ during the acute hypoxic response tests (P = 0.88). Acute hypercapnic response

There were no significant differences in the SBP, DBP, MAP and CVR response to acute hypercapnia. Table 2 displays the values for each response on each day of measurement. Discussion Figure 2. Resting systolic (SBP), diastolic (DBP), and mean arterial pressure (MAP) prior to acute testing Below each figure the time effect is displayed. Calipers display the P-value following post hoc analysis comparing Day 4 and Day 8 (recovery) to Baseline.

Main findings

We have demonstrated in an experimental human model that exposure of healthy humans to IH, in a mode similar C 2009 The Authors. Journal compilation C 2009 The Physiological Society

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to that experienced by patients with OSA, increased resting arterial blood pressure. Furthermore, IH increased the pressor response to hypoxia, increased the cerebral vascular resistance response to hypoxia, and decreased nitric oxide derivatives. There were no associated changes in the cardiovascular and cerebral vascular responses to hypercapnia. These findings suggest that the increased risk of cardiovascular and cerebral vascular disease in patients with OSA may be due, at least in part, to the effects of IH on the vasculature. Intermittent hypoxia and regulation of arterial blood pressure

There is a strong epidemiological association between OSA and hypertension that is independent of confounding factors such as age and weight (Somers et al. 2008). This association has been attributed to IH. Rats exposed to two 12 s bouts of hypoxia per minute (nadir F I,O2 = 3–5%), 7 h per day for 35 days develop systemic hypertension (Fletcher et al. 1992c). In these experiments, MAP increased by 13.5 mmHg. In our study with human subjects, using a shorter exposure to IH (4 vs. 35 days)

and with fewer daily bouts of hypoxia (1 bout every 4 min vs. 8 bouts every 4 min) we found a 4 mmHg increase in MAP (Fig. 2). Further, we found that the increase in MAP was reversible since it returned toward baseline 4 days after exposure to IH was discontinued. IH has a profound effect on MAP, and is likely to be the mechanism by which hypertension develops in patients with OSA. This suggests that treatment of OSA could have significant implications for stroke and coronary heart disease as a 5–6 mmHg reduction in DBP reduces the risk of stroke by 33–50% and the risk of coronary heart disease by 4–22% (Collins et al. 1990). IH may increase systemic arterial pressure by augmenting peripheral chemoreflex sensitivity, thereby enhancing sympathetic outflow to systemic vessels and increasing vascular tone and total peripheral vascular resistance (Foster et al. 2007b). Peripheral vascular resistance can also be increased by renal sympathetic nerve activity, which leads to greater production of angiotensin-II, a potent vasoconstrictor (Hackenthal et al. 1990). Although these pathways have been well described in animal studies of IH (Fletcher et al. 1992a,b,c, 1999, 2002; Fletcher, 2000), it has not been confirmed whether

Figure 3. Example of a 6 h exposure to intermittent hypoxia in a single subject Left panel displays the first 30 min of intermittent hypoxia. The middle panel shows the following 5 h of exposure and the right panel displays the final 30 min of exposure. The traces from top to bottom are: end-tidal partial pressure of oxygen (P ET,O2 ), arterial oxyhaemoglobin saturation (Sa,O2 ), end-tidal partial pressure of carbon dioxide (P ET,CO2 ), mean arterial pressure (MAP), and middle cerebral artery peak blood flow velocity (V¯ P ). C 2009 The Authors. Journal compilation C 2009 The Physiological Society

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Figure 4. Overlay of morning and afternoon intermittent hypoxia cycles on the first and fourth days of exposure Continuous black line and circle: overlay of the first six cycles of intermittent hypoxia (IH) on Day 1. Dotted black line and triangle: overlay of the last six cycles of IH on Day 1. Continuous grey line and circle: overlay of first six cycles of IH on Day 4. Dotted grey line and triangle: overlay of last six cycles of IH on Day 4. Each symbol represents a 15s mean ± S.E.M. centred on 60 s, 120 s, 180 s and 240 s for all subjects (n = 10). Brackets on the right side of panel D refer to a Day 1 versus Day 4 effect. Brackets on the right side of panels E and F refer to a morning vs. afternoon effect. Definition of abbreviations: P ET,O2 , end-tidal partial pressure of oxygen; P ET,CO2 , end-tidal partial pressure of carbon dioxide; Sa,O2 , arterial oxyhaemoglobin saturation; V¯ P , middle cerebral artery mean peak blood flow velocity; MAP, mean arterial pressure; CVR, cerebral vascular resistance. Comparisons are reported for the 15 s values at 120 s (end-period of normoxia) and 240 s (end-period of hypoxia). C 2009 The Authors. Journal compilation C 2009 The Physiological Society


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Table 2. The hypercapnic sensitivities for the percent change in systolic (SBP), diastolic (DBP), mean arterial pressure (MAP), and cerebrovascular resistance (CVR) for each day of intermittent hypoxia

mmHg−1 )

SBP/P ET,CO2 (% DBP/P ET,CO2 (% mmHg−1 ) MAP/P ET,CO2 (% mmHg−1 ) CVR/P ET,CO2 (% mmHg−1 )

Baseline

Day 1

Day 2

Day 3

Day 4

Day 8

0.96 ± 0.11 0.88 ± 0.10 0.91 ± 10.0 −2.82 ± 0.16

0.95 ± 0.23 0.94 ± 0.25 0.98 ± 0.20 −2.84 ± 0.24

0.78 ± 0.22 0.83 ± 0.20 0.84 ± 0.18 −2.56 ± 0.25

0.95 ± 0.19 1.03 ± 0.21 1.00 ± 0.18 −2.50 ± 0.18

1.31 ± 0.27 1.19 ± 0.25 1.19 ± 0.22 −2.94 ± 0.18

1.10 ± 0.22 1.05 ± 0.13 1.05 ± 0.11 −2.54 ± 0.26

they play a role in humans. Previous human studies have shown a positive relationship between peripheral chemosensitivity and the pressor response to hypoxia following exposure to IH (Katayama et al. 2001; Foster et al. 2005). Furthermore, IH increases chemoreflex control of sympathetic outflow (Lusina et al. 2006). We have extended these findings by demonstrating that prolonged bouts of IH, which are more similar to the hypoxaemia experienced by OSA patients, increases the

pressor response to hypoxia (Fig. 5) and this suggests that IH is a likely mechanism for increased risk of hypertension in OSA. We observed a 10 ± 4% increase in MAP during the first day of exposure to IH (Day 1) which progressed to 21 ± 10% by Day 4 (Fig. 4). A similar finding has been reported by Leuenberger et al. (2005) who observed that 30 min of intermittent breath-holds primed with a hypoxic gas mixture and held for 20 s out of each minute

Figure 5. Regression lines for systolic (SBP), diastolic (DBP), and mean arterial pressures (MAP) and the cerebrovascular resistance (CVR) plotted against arterial oxyhaemoglobin saturation (Sa,O2 ) for baseline and day 4 All regression lines were not coincident (P < 0.01) and not parallel (P < 0.001). The slope of each line and the r 2 value is shown for each plot. C 2009 The Authors. Journal compilation C 2009 The Physiological Society

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transiently increased blood pressure and sympathetic activity. Foster et al. (2005) also reported that during 1 h of IH (5 min 12% O 2 alternating with 5 min 21% O 2 ), MAP was less during the first 5 min of hypoxia compared with the last 5 min of hypoxia. Our findings are consistent with these previous investigations, which suggest that the pressor response to hypoxia increases across a single exposure to IH, and adds to the previous studies by indicating that on-going exposure to IH further potentiates this response. The pressor response to hypercapnia was not changed by exposure to IH. This finding was not surprising since the sympathetic nervous system response to hypercapnia differs from the response to hypoxia and has not been associated with long standing sympathetic outflow even following removal of the stimulus (Xie et al. 2001).

Intermittent hypoxia and cerebral vascular regulation

There is also an epidemiological association between OSA and the risk of stroke (Arzt et al. 2005; Yaggi et al. 2005) that may be caused by alteration in cerebral vascular regulation due to IH. Several recent studies demonstrate that cerebral blood flow regulation is altered in OSA patients (Placidi et al. 1998; Foster et al. 2007a; Urbano et al. 2008). We have recently found that the cerebral blood flow response to hypoxia is blunted in OSA patients and that it returns to normal after 4–6 weeks of continuous positive airway pressure (CPAP) therapy (Foster et al. 2007a). Such a reduction in the cerebral vasodilator response to hypoxia could be due both to an increase in vasoconstrictor activity as described above, and also to reduced production of nitric oxide, a vasodilator that is thought to be involved in basal vascular tone and the vasodilator response to hypoxia (Ray et al. 2002). Nitric oxide production is reduced by the increased oxidative stress associated with IH (Clapp et al. 2004). Indeed, plasma levels of nitric oxide by-products are decreased in OSA patients and increase following correction of IH by CPAP therapy (Ip et al. 2000; Schulz et al. 2000). Our results indicate that the cerebral vasodilator response to hypoxia is decreased by exposure to IH as reflected by an increase in cerebral vascular resistance following the fourth day of IH compared to baseline (Fig. 5). Previous studies of human subjects exposed to IH also support this hypothesis (Foster et al. 2005; Querido et al. 2008). The reduced cerebral vasodilator response to hypoxia following exposure to IH may be due to inactivation of nitric oxide and endothelial dysfunction. Animal studies support the hypothesis that cerebral vessels have a reduced vasodilator response following exposure to IH and that this is likely to be due to reduced bioavailability of nitric oxide (Tahawi et al. 2001; Phillips et al. 2004).

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This hypothesis is supported by our findings that NOx, an indicator of nitric oxide production, was reduced by 55% following exposure to IH (Table 1). Furthermore, the fall in nitric oxide associated with IH was reversible since it returned to baseline levels 4 days after exposure to IH finished. This finding is consistent with previous human studies on OSA where nitric oxide increased by a similar magnitude following correction of OSA, and associated hypoxaemia, with CPAP therapy (Ip et al. 2000). Exposure to IH was associated with significant reductions in P ET,CO2 secondary to increased peripheral chemoreceptor sensitivity and ventilation. This reduction in P CO2 might be expected to decrease resting cerebral blood flow due to the cerebral vasoconstrictive effect of hypocapnia. Cerebral blood flow was reduced in the morning immediately prior to IH exposure on Day 4. However, resting cerebral blood flow, measured in the afternoon prior to acute hypoxia testing, was not different across the protocol despite significantly reduced P ET,CO2 . The reason for this is unclear and warrants further investigation. The cerebral circulation did manifest increased resistance during the afternoons of Day 1 and Day 4. CVR was increased by 10 ± 6% on Day 1 and by 15 ± 9% on Day 4 (see Fig. 4). This finding is due to increased MAP and not due to decreases in P ET,CO2 , as the level of P ET,CO2 did not decrease throughout a single day of exposure to IH (Fig. 4). In addition, cerebral autoregulation seems to be negatively affected by exposure to intermittent hypoxia. The higher cerebral blood flow velocity observed in the afternoon on Day 4 demonstrates this altered cerebral autoregulation (see Fig. 4). This may place the cerebral circulation at an increased risk for cerebral vascular events (e.g. haemorrhage) due to the combined effect of systemic hypertension and loss of cerebral autoregulation. The cerebral blood flow response to hypercapnia was not altered by exposure to IH. This finding may be explained by differences in the mechanism for cerebral vasodilatation induced by hypercapnia compared to that of hypoxia. While the precise mechanisms for hypercapnic vasodilatation are not fully understood, it appears to be mediated by reduced extracellular and/or intracellular pH and by the presence of nitric oxide and prostacyclin (Hsu et al. 1993; Wang et al. 2003; Heintz et al. 2005). Since only the presence of nitric oxide and prostacyclin are required and their graded release does not appear to be necessary for the hypercapnic vasodilatory response, this may be one explanation for which the cerebral blood flow response to hypercapnia was unaffected by IH (Hsu et al. 1993; Heintz et al. 2005). In addition, a normal cerebral blood flow response to hypercapnia was recently reported in severe OSA patients without coexisting cardiovascular disease (Foster et al. 2009). C 2009 The Authors. Journal compilation C 2009 The Physiological Society

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Comparison of IH exposure in our model to that in OSA

Our model of IH was designed to simulate the arterial oxygen desaturation profile observed in patients with OSA. Moderately severe OSA is defined as an AHI of 15 to 30 events per hour of sleep (American Academy of Sleep Medicine, 1999). Apnoea is defined as complete cessation of airflow for 10 s or more, whereas hypopnoea is defined as a critical reduction in airflow for 10 s or more that is accompanied by a fall in S a,O2 of 4% or more and/or an arousal from sleep. In our study we measured the ODI, which is defined as the number of times the S a,O2 decreased by 4%. The ODI experienced by our subjects (14.8 ± 0.1) equates to the oxygen desaturation profile experienced by patients with mild to moderate OSA. Furthermore, we observed a mean minimum S a,O2 of 83.3 ± 0.5% and a mean maximum S a,O2 of 95.9 ± 0.1%. These results are comparable to those from studies on patients with OSA (Choi et al. 2000), and also demonstrates that our model induced significant oscillation in oxygen saturation and consequent exposure to IH. Nevertheless, our model does have some limitations. First, the duration of each hypoxic episode was 2 min whereas the average duration of an apnoea in patients with OSA is about 20 s (O’Connor et al. 2000). Second, our intermittent hypoxia paradigm did not control P CO2 , which fell by approximately 4 mmHg (Figs 3 and 4). Typically, obstructive apnoeas are associated with a 6 mmHg increase in end-expiratory P CO2 (Klingelhofer et al. 1992). While this may have implications for cerebral vascular control, exposure to hypercapnia may not be critical for the development of cardiovascular and cerebrovascular disease. Previous research using animal models have shown that the level of arterial P CO2 during exposure to IH does not alter the development of hypertension. However, hypercapnic IH may cause greater sympathetic activation than hypocapnic hypoxia (Tamisier et al. 2004). Despite these limitations, we believe that our model of IH is innovative and is an effective experimental paradigm with which to study the direct effects of IH on the cardiovascular system. This model specifically evaluates the effects of IH without potential confounding factors, such as sleep fragmentation, arousals, intrathoracic pressure swings, and coexisting cardiovascular disease, all of which are common in studies of patients with OSA. Typically, cerebral blood flow increases during apnoea with a steep rise at the end of apnoea (Balfors & Franklin, 1994). As breathing resumes, cerebral blood flow drops dramatically before returning toward baseline values (Balfors & Franklin, 1994). MAP also rises during obstructive apnoeas and drops at the resumption of breathing followed by a progressive increase toward baseline (Balfors & Franklin, 1994). Another limitation C 2009 The Authors. Journal compilation C 2009 The Physiological Society

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of our model is that the temporal changes in cerebral blood flow and MAP do not follow the typical dynamics that have been described in patients with OSA (Fig. 4). We observed a dramatic drop in cerebral blood flow as hypoxia is initiated which is likely to be due to decreases in P ET,CO2 associated with the ventilatory response to hypoxia. Cerebral blood flow increased progressively, likely to be due to the ensuing hypoxia. Once hypoxia ended, cerebral blood flow continued to increase as the P ET,CO2 returned toward baseline. The temporal pattern of changes in MAP associated with IH was more similar to that described in OSA. It increased during the hypoxic period, dropped rapidly during normoxia, and slowly returned toward baseline. Future studies should aim to reduce the length of each bout of hypoxia and maintain isocapnia (or induce a mild hypercapnia) in order to replicate OSA more precisely. We studied the effects of 4 days of IH on cardiovascular and cerebrovascular regulation. Using a well-controlled experimental human model, we found that an IH profile similar to that seen in OSA leads to cardiovascular and cerebrovascular dysregulation, manifested by increased resting blood pressure, an enhanced pressor response to hypoxia, decreased nitric oxide production and reduced cerebrovascular response to hypoxia. These new findings advance our current understanding of the pathophysiological mechanisms for the development of cardiovascular and cerebrovascular disease in patients with OSA. References American Academy of Sleep Medicine (1999). Sleep-related breathing disorders in adults: recommendations for syndrome definition and measurement techniques in clinical research. The Report of an American Academy of Sleep Medicine Task Force. Sleep 22, 667–689. Ainslie PN & Poulin MJ (2004). Ventilatory, cerebrovascular, and cardiovascular interactions in acute hypoxia: regulation by carbon dioxide. J Appl Physiol 97, 149–159. Arzt M, Young T, Finn L, Skatrud JB & Bradley TD (2005). Association of sleep-disordered breathing and the occurrence of stroke. Am J Respir Crit Care Med 172, 1447–1451. Balfors EM & Franklin KA (1994). Impairment of cerebral perfusion during obstructive sleep apneas. Am J Respir Crit Care 150, 1587–1591. Choi S, Bennett LS, Mullins R, Davies RJ & Stradling JR (2000). Which derivative from overnight oximetry best predicts symptomatic response to nasal continuous positive airway pressure in patients with obstructive sleep apnoea? Respir Med 94, 895–899. Clapp BR, Hingorani AD, Kharbanda RK, Mohamed-Ali V, Stephens JW, Vallance P & MacAllister RJ (2004). Inflammation-induced endothelial dysfunction involves reduced nitric oxide bioavailability and increased oxidant stress. Cardiovasc Res 64, 172–178.

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Author contributions Conception and design: G.E.F., P.J.H., M.J.P. Data collection: G.E.F., J.V.B., V.P., C.T.C.D., S.B.A. Analysis and interpretation: G.E.F., J.V.B., V.P., C.T.C.D., P.J.H., S.B.A., M.J.P. Article drafting and revisions: G.E.F., J.V.B., V.P., C.T.C.D., P.J.H., S.B.A., M.J.P.

Acknowledgements We thank our dedicated subjects for their participation in this study. We are grateful to Dr D. Roach for his assistance with computer programming. Support was provided by the Alberta Heritage Foundation for Medical Research (AHFMR; Establishment Grant: M.J.P.), the Heart and Stroke Foundation of Alberta, Northwest Territories & Nunavut (Special Patient-Centered Grant-in-Aid: P.J.H. and M.J.P.), and the Canadian Foundation for Innovation (New Opportunities Fund: M.J.P.). G.E.F. is a Canadian Institutes of Health Research Strategic Training Fellow in TORCH (Tomorrow’s Research Cardiovascular Health Professionals), and was supported by doctoral research awards from the AHFMR and the Heart and Stroke Foundation of Canada. J.V.B. was a Hotchkiss Brain Institute post-doctoral fellow. V.P. was an AHFMR post-doctoral fellow. C.T.C.D. was a CIHR training fellow in TORCH and was funded by a master’s research award from the CIHR. S.B.A. is supported by the Kidney Foundation of Canada. M.J.P. is an AHFMR Senior Medical Scholar.