The Journal of Physiology

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1 J Physiol (2014) pp Impact of hypocapnia and cerebral perfusion on orthostatic tolerance NiaC.S.Lewis 1,AnthonyR.Bain 1,DavidB.MacLeod 2,KevinW.Wildfong 1, Kurt J. Smith 1, Christopher K. Willie 1, Marit L. Sanders 3, Tianne Numan 3, Shawnda A. Morrison 1,4,GlenE.Foster 1, Julian M. Stewart 5 and Philip N. Ainslie 1 1 Centre for Heart, Lung and Vascular Health, University of British Columbia, Okanagan, Canada 2 Department of Anesthesiology, Duke University Medical Center, Durham, NC, USA 3 MIRA, University of Twente, Enschede, The Netherlands 4 Jozef Stefan Institute, Ljubljana, Slovenia 5 New York Medical College, New York, USA The Journal of Physiology Key points Vasovagal syncope (a common form of fainting) is frequently associated with excessive breathing and leads to reductions in carbon dioxide (hypocapnia) and cerebral hypoperfusion. The prevention of hypocapnia during orthostatic stress has been shown to improve orthostatic tolerance, but it still remains to be quantified in a larger population, with a more sustained orthostatic stress. Resting brain blood flow has been shown to impact orthostatic tolerance; however, the importance of resting brain blood flow per se in the pathophysiology of vasovagal syncope has not been clearly explicated. Our findings show that cerebral hypoperfusion either at rest or induced by hypocapnia at pre-syncope do not impact on orthostatic tolerance, probably due to a compensatory increase in oxygen extraction of the brain. Abstract We examined two novel hypotheses: (1) that orthostatic tolerance (OT) would be prolonged when hyperventilatory-induced hypocapnia (and hence cerebral hypoperfusion) was prevented; and (2) that pharmacological reductions in cerebral blood flow (CBF) at baseline would lower the CBF reserve, and ultimately reduce OT. In study 1 (n = 24; aged 25 ± 4years) participants underwent progressive lower-body negative pressure (LBNP) until pre-syncope; end-tidal carbon dioxide (P ET,CO2 ) was clamped at baseline levels (isocapnic trial) or uncontrolled. In study 2 (n = 10; aged 25 ± 4 years), CBF was pharmacologically reduced by administration of indomethacin (INDO; 1.2 mg kg 1 ) or unaltered (placebo) followed by LBNP to pre-syncope. Beat-by-beat measurements of middle cerebral artery blood flow velocity (MCAv; transcranial Doppler), heart rate (ECG), blood pressure (BP; Finometer) and end-tidal gases were obtained continuously. In a subset of subjects arterial-to-jugular venous differences were obtained to examine the independent impact of hypocapnia or cerebral hypoperfusion (following INDO) on cerebral oxygen delivery and extraction. In study 1, during the isocapnic trial, P ET,CO2 was successfully clamped at baseline levels at pre-syncope (38.3 ± 2.7 vs ± 2.5 mmhg respectively; P = 0.). In the uncontrolled trial, P ET,CO2 at pre-syncope was reduced by 10.9 ± 3.9 mmhg (P 0.001). Compared to the isocapnic trial, the decline in mean MCAv was 15 ± 4cms 1 (35%; P 0.001) greater in the uncontrolled trial, yet the time to pre-syncope was comparable betweentrials(544± 130 vs. 572 ± 180 s; P = 0.30). In study 2, compared to placebo, INDO reduced resting MCAv by 19 ± 4cms 1 (31%; P 0.001), but time to pre-syncope remained similar between trials (placebo: 1123 ± 138 s vs. INDO: 1175 ± 212 s; P = 0.53). The brain extracted more oxygen in face of hypocapnia (34% to 53%) or cerebral hypoperfusion (34% to DOI: /jphysiol

2 5204 N. C. S. Lewis and others J Physiol %) to compensate for reductions in delivery. In summary, cerebral hypoperfusion either at rest or induced by hypocapnia at pre-syncope does not impact OT, probably due to a compensatory increase in oxygen extraction. (Received 8 July 2014; accepted after revision 1 September 2014; first published online 12 September 2014) Corresponding author N. C. S. Lewis: Centre for Heart, Lung and Vascular Health, University of British Columbia, Okanagan Campus, School of Health and Exercise Sciences, 111 Health Science Centre, 1088 Discovery Avenue, Kelowna, British Columbia, Canada. Nia.Lewis@ubc.ca Abbreviations ANOVA, analysis of variance; BP, blood pressure; CA, cerebral autoregulation; CBF, cerebral blood flow; CDO 2, cerebral delivery of oxygen; CO, cardiac output; CVCi, cerebrovascular vascular conductance index; CVRi, cerebrovascular resistance index; DBP, diastolic blood pressure; DMCAv, diastolic middle cerebral artery velocity; DPCAv, diastolic posterior cerebral artery velocity; gcbf, global CBF; HR, heart rate; HUT, head up tilt; INDO, indomethacin; LBNP, lower body negative pressure; MAP, mean arterial blood pressure; MCA CVRi, middle cerebral artery cerebral vascular resistance index; MCAv, middle cerebral artery velocity; OT, orthostatic tolerance; P a,co2,arterial carbon dioxide; PCA CVRi, posterior cerebral artery cerebral vascular resistance index; P ET,CO2, end-tidal carbon dioxide; P ET,O2, end-tidal oxygen; POTS, postural tachycardia syndrome; PCAv, posterior cerebral artery; SBP, systolic blood pressure; SD, standard deviation; SMCAv, systolic middle cerebral artery velocity; SPCAv, systolic posterior cerebral artery velocity; SV, stroke volume; TPRi, total peripheral resistance index. Introduction Syncope is a transient, sudden loss of consciousness and postural tone, which is often but not always preceded by pre-syncopal symptoms (e.g. visual disturbances, dizziness, nausea (Stewart, 2013). Postural vasovagal syncope is the most common form and usually occurs in the presence of orthostatic hypotension; irrespective of the condition provoking syncope onset, loss of consciousness is fundamentally due to cerebral hypoperfusion and inadequate cerebral oxygenation (Van Lieshout et al. 2003; Thomas et al. 2009b). Currently, the exact pathophysiological mechanisms influencing cerebral hypoperfusion and syncope onset are unknown, but are multifactorial. Vasovagal syncope is frequently associated with hyperventilation, which has been reported in the pathophysiology of syncope (Saltzman et al. 1963; Grubb et al. 1991; Martinon Torres et al. 2001; Van Lieshout et al. 2003; Porta et al. 2008). Furthermore, hypotension induced by postural (LeLorier et al. 2003; Convertino et al. 2009) and pharmacological (Stewart et al. 2011) methods in healthy individuals, has been shown to stimulate hyperventilation and the development of hypocapnia, and is thought to be evoked at least in part by ventilatory baroreflex interactions (Stewart et al. 2011). The cerebral vasculature is highly sensitive to changes in the partial pressure of arterial carbon dioxide (P a,co2 ), such that hypocapnia causes increased cerebral vascular resistance and decreased cerebral blood flow (CBF) (Willie et al. 2012). Hypocapnia induced cerebral hypoperfusion has been associated with orthostatic intolerance (Novak et al. 1998; Serrador et al. 2006). There is evidence to suggest that hyperventilatory-induced hypocapnia during head up tilt (HUT) causes vasodepression, i.e. reduced vascular resistance and hence blood pressure (BP; Thijs et al. 2008), which could ultimately impact orthostatic tolerance (OT). In contrast, in postural tachycardia syndrome (POTS), there is evidence that sustained hypocapnia and cerebral ischaemia produce sympathoexcitation, tachycardia, and consequent elevations in blood pressure (Del Pozzi et al. 2014). However, only a few studies have noted the effect of P a,co2 on OT time. Breathing 5% CO 2 has been shown to improve OT by 10%, and it was speculated that this improvement in OT was attributed to a delayed reduction in CBF by way of elevated resting CBF (although CBF was not measured; Howden et al. 2004). Blaber et al. (2001) found that maintaining end-tidal carbon dioxide (P ET,CO2 ) at baseline levels during upright tilt did not eliminate cerebral hypoperfusion; however, in four out of the five individuals who experienced pre-syncope onset, OT was improved. This improvement in tolerance time was associated with a higher resting middle cerebral artery velocity (MCAv). Given that the orthostatic challenge adopted by Blaber et al. (2001) consisted of a series of six 10-min 90 deg HUTs, pre-syncope was not induced in a controlled manner; therefore, whether the prevention of hypocapnia during orthostatic stress improves OT, still remains to be quantified in a larger sample size with more sustained orthostatic stress and control of P ET,CO2. Ithasbeennotedoveryearsagothatthedevelopment of syncope and symptoms of cerebral ischaemia develop when CBF is reduced below a critical threshold of 35% below resting values, irrespective of a wide variation in BP ( 19% to 64%;e.g.Finnertyet al. 1954). McHenry et al. (1961) in an elderly and hypertension population associated this critical level of cerebral hypoperfusion with a reduction in cerebral oxygen delivery. As highlighted above, CO 2 supplementation has been show to improve OT which was suggested to be mediated via an elevation

3 J Physiol Syncope: role of hypocapnia and cerebral hypoperfusion 5205 in resting MCAv and thus a heightened cerebral reserve (i.e. difference between baseline CBF and the flow evident at syncope; Howden et al. 2004). Somewhat consistent with this notion, others have reported a 22% decline in OT in the morning compared to the evening, partly due to a lower CBF reserve as reflected by a 15% lower morning resting MCAv (Lewis et al. 2010). These findings suggest cerebral hypoperfusion at rest can negatively impact OT, yet the importance of resting CBF per se in the pathophysiology of OT, has not been clearly explicated. More specifically, and of relevance to this study, it is currently unknown how the level of hypocapnia associated with hyperventilation preceding syncope, and therefore associated cerebral hypoperfusion, influences cerebral oxygen delivery independently of the development of hypotension. In two main investigations, we aimed to independently examine the effects of hyperventilatory-induced hypocapnia and cerebral hypoperfusion on OT. Study 1 aimed to examine the contribution of hypocapnia-induced cerebral hypoperfusion on OT. To address this question, P ET,CO2 was clamped at baseline values via an end-tidal forcing approach during progressive lower body negative pressure (LBNP) to pre-syncope. We hypothesized that tolerance time to progressive LBNP would be longer when hyperventilatory-induced hypocapnia was prevented.study2investigatedtheinfluenceofcerebralhypoperfusion on OT. We achieved this by pharmacological manipulation of resting CBF with administration of indomethacin (INDO; a non-selective cyclooxygenase 1 and 2 inhibitor). Indomethacin at clinically relevant doses (as given in this study) lowers CBF by 25 % (Markus et al. 1994; Bruhn et al. 2001) without concomitant changes in metabolic rate (Hohimer et al. 1985; Kraaier et al. 1992), plasma catecholamines (Wennmalm et al. 1984) or arterial CO 2 (P a,co2 ;Fanet al. 2010, 2011). We hypothesized that the administration of INDO would lower the CBF reserve, and ultimately reduce OT. In a subset of subjects, arterial-to-jugular venous differences were obtained to examine the independent impact of hypocapnia (matched to individual levels measured in study 1) and cerebral hypoperfusion (prior to and following INDO as per study 2) on cerebral oxygen delivery and extraction. Methods Ethical approval and screening Studies 1 and 2 were approved by the Human Ethics Committee of the University of British Columbia, and conformed to the standards set by the Declaration of Helsinki. All subjects provided written informed consent. Participants were non-smokers, had no previous history of cardiovascular, cerebrovascular or respiratory diseases, and were not taking any cardiovascular medications. All participants reported no past episodes of syncope and/or related symptoms in their normal daily lives within the last 6 months. Room temperature was maintained at C, and experimental testing began after 12-h abstinence from alcohol, caffeine, and strenuous exercise, and at least a 4-h fast. Experimental testing took place between h and h. Female participants were tested in the early follicular phase (day 1 7) of the menstrual cycle day 1 being the first day of menstruation, or during menstruation of the pill withdrawal phase (day 2 7). All participants underwent a familiarization session of the experimental procedures and protocol on a separate day ( 24 h) prior to their initial assessment. Experimental design Study 1. Twenty-four (12 males; 12 females) healthy participants with a mean ± standard deviation (SD) age of 25 ± 4 years, body mass 69 ± 11 kg, height 178 ± 8 cm, and body mass index 23.2 ± 3.3kgm 2 participated in this study. The design was a two-trial, counterbalanced experiment involving progressive (LBNP until pre-syncope, with P ET,CO2 and end-tidal oxygen (P ET,O2 ) clamped at baseline levels (isocapnia condition) or uncontrolled (poikilocapnic condition). Following instrumentation and 20 min of supine rest, participants engaged in a supine LBNP protocol. Lower body suction was applied in 20 mmhg incremental steps, each lasting 2 min, until 100 mmhg was reached, after which 100 mmhg was continued until pre-syncope onset (see Determination of pre-syncope and orthostatic tolerance below). Following 1 h and 20 min of supine rest, participants underwent the LBNP again under the different condition. Study 1B (hypocapnic challenge). A subset of the recruited participants (n = 5; 28 ± 3 years) underwent an additional protocol to examine the response of hypocapnia, independent of systemic hypotension on CBF regulation. Invasive arterial jugular venous differences and volumetric CBF measures were made to directly calculate alterations in oxygen delivery and extraction. A 20 gauge peripheral intravenous catheter and 20 gauge radial artery catheter were inserted following local anaesthesia with 1% lidocaine. Ultrasound was used to identify the internal jugular vein and assist with the cannulation of the vein. A jugular bulb catheter (Edwards, Seattle, WA, USA) was placed in the right internal jugular vein by the Seldinger technique and directed cephalad under sterile conditions. Following 15 min of supine rest, individuals were instructed to hyperventilate at their desired combination of frequency and tidal volume to induce a steady-state (>5 min) level hypocapnia (P ET,CO2 : 23 ± 5 mmhg) equivalent

4 5206 N. C. S. Lewis and others J Physiol to the level experienced in the syncope trial (study 1). Intra-arterial blood pressure and velocity in the middle (MCAv) and posterior cerebral artery (PCAv) were measured continuously (see Measurements section for more details). Global CBF (gcbf) was estimated via diameter and blood velocity recordings in the left internal carotid artery and right vertebral artery obtained using a 10 MHz multifrequency linear array probe attached to a high-resolution ultrasound machine (Terason 3000, Teratech,Burlington,MA, USA; see Willie et al. (2012) for further details regarding ultrasound data collection and analysis). Imaging of the extracranial arteries commenced when end-tidal clamping was stable. Arterial and venous blood gas samples from the radial artery and jugular catheter, respectively, were drawn simultaneously near the end of the step change in P a,co2 into a preheparinized syringe, and immediately analysed. Following standardised calibration, all blood samples were analysed using an arterial blood-gas analysing system (ABL-90 Co-Ox, Radiometer, Copenhagen, Denmark) for P O2, P CO2 and arterial oxygen saturation. Global CBF, arterial oxygen delivery, venous content of oxygen, cerebral delivery of oxygen (CDO 2 ), cerebral oxygen extraction (CO 2 extraction) were calculated (see Ainslie et al for detailed calculations). Study 2. In study 2, pharmacological manipulation of CBF, independent of changes in P ET,CO2, was achieved by ingesting INDO (1.2 mg kg 1 ; or a placebo) 90 min before undergoing a progressive LBNP protocol to pre-syncope. Ten healthy males with a mean ± SD age of 25 ± 4years, body mass 71 ± 10 kg, height 173 ± 10 cm, and body mass index 23.2 ± 3.3kgm 2,tookpartinthis randomized, placebo controlled experiment. Participants attended the laboratory on two occasions, occurring at the same time of the day (separated by 24 h) and followed the same experimental procedures. Following capsule ingestion, instrumentation and 20 min of supine rest, participants engaged in a supine LBNP protocol: LBNP was applied in 6 min stages at 25,, 75 and 100 mmhg until pre-syncope (see Determination of pre-syncope and orthostatic tolerance below). This LBNP protocol differed slightly from study 1 in order to elicit a more sustained orthostatic stimuli to ensure steady state was achieved at each level so that the cardiorespiratory and cerebrovascular reflexes could fully develop. This is unlikely to be a major limitation given the within-experimental comparison. Study 2B. To examine the effect of INDO on CBF regulation at rest, invasive arterial jugular venous differences were measured (n = 4; 26 ± 6 years; same procedures as detailed in study 1B) to directly calculate alterations in P O2, P CO2 and arterial oxyhaemoglobin saturation, arterial oxygen delivery, CDO 2, and O 2 extraction. Following 15 min of supine rest prior to and 90 min following INDO ingestion (1.2 mg kg 1 ), arterial and venous blood gas samples were simultaneously drawn from the radial artery and jugular catheter respectively, into a preheparinized syringe and analysed immediately. Determination of pre-syncope and orthostatic tolerance In line with previous research (Levine et al. 1994; Thomas et al. 2009b;Lewiset al. 2010, 2012), pre-syncope and OT was defined by a sustained drop in systolic BP <80 mmhg for more than 10 s or on participants request due to one or more subjective presyncopal symptoms becoming intolerable (feelings of dizziness, nausea, faintness, visual disturbances, hearing disturbances, and fatigue). Pre-syncopal symptoms were recorded using a validated questionnaire (Thomas et al. 2009b) immediately after the cession of the experimental protocol. Participants were asked to rate their symptoms based upon a visual scale from1to10(1beingnosymptomsto10beingintolerable), for the period related to the end of the challenge or at the point of pre-syncope. Orthostatic tolerance was determined as the time elapsed before termination of the LBNP. Measurements Beat-to-beat measurements of MCAv and PCAv, were acquired using a 2-MHz pulsed transcranial Doppler ultrasound system (Spencer Technologies, Seattle, WA, USA). Using search methods described elsewhere (Willie et al. 2011), the Doppler probe was maintained in position, at a fixed angle, using a commercially available fixation headframe (Marc 0; Spencer Technologies). Stroke volume (SV) and cardiac output (CO) were calculated from the BP waveform obtained from finger photoplethysmography using the Modelflow method, incorporating age, sex, height, and weight (BeatScope 1.0 software; TNO TPD; Biomedical Instruments, Amsterdam, The Netherlands) (Wesseling et al. 1993). Total peripheral resistance index (TPRi) was calculated from mean arterial BP (MAP/CO). Cerebrovascular resistance index (CVRi) was calculated as MAP/MCAv. Cerebrovascular vascular conductance index (CVCi) was calculated as MCAv or PCAv/MAP. For measurement of P ET,CO2 and P ET,O2, subjects breathed through a mouthpiece and two-way non-rebreathing valve. Respired gas pressures were sampled at the mouth by securing a calibrated online gas analyser (model ML206, AD Instruments, Colorado Springs, CO, USA) into the mouthpiece and respiratory flow was measured at the mouth using a pneumotachograph (model HR 800L, HansRudolph, Shawnee, KS, USA).

5 J Physiol Syncope: role of hypocapnia and cerebral hypoperfusion 5207 End-tidal forcing for study 1 was achieved as described in depth elsewhere (Bain et al. 2013; Foster et al. 2014) In brief, on a breath-by-breath bases, P ET,CO2, P ET,O2, inspiratory and expiratory tidal volume were determined online using specifically designed software (LabView, Austin, TX, USA), and P ET,O2 and P ET,O2 were controlled by a portable end-tidal forcing system (Air- Force, GE Foster, Kelowna, BC, Canada). This system uses independent gas solenoid valves for oxygen, carbon dioxide, and nitrogen and controls the volume of each gas being delivered to the inspiratory reservoir through a mixing-and-humidification chamber. With use of feedback information regarding P ET,CO2, P ET,O2, and inspiratory and expiratory tidal volume, the system prospectively targets the inspirate to bring end-tidal gas to the desired level. Gas control was fine-tuned using a feedback control and error reduction algorithms. Clamped P ET,CO2 and P ET,O2 levels were determined as the values measured during the last 5 min of baseline measurements. The experimental protocol during the end-tidal clamped trial did not start until 3 min into clamping to ensure that P ET,CO2, P ET,O2, MCAv and PCAv had reached steady state. All data were sampled continuously at 200 Hz using an analogue digital converter (PowerLab/16SP ML795; ADInstruments, Colorado Springs, CO, USA) interfaced with a computer and displayed in real time during testing. Data were stored for subsequent off-line analysis using the commercially available LabChart software (version 7.3.7; ADInsturments). Baseline values represent a 1-min average prior to LBNP, and pre-syncope values represent a 10 s average prior to the termination of the test protocol. Statistical analysis All data were analysed using SPSS (version 21, Surrey, UK) and expressed as mean ± SD. Statistical significance was defined as P Study 1. Trial differences in OT were determined using a one-tailed paired t test. Two-way repeated measures ANOVA was used to examine the interaction between any time point (baseline vs. pre-syncope) and experimental condition (unclamped vs. clamped trials). In the event of any significant interaction, paired t tests were then used (provided the data was normally distributed). A two-way repeated measures ANOVA was used to explore the interaction between sex (males vs. female) on OT and the time course change (baseline vs. syncope). In study 1B, within-study differences between normocapnia and hypocapnia were assessed via a paired t test, provided data were normally distributed. Study 2. A two-way repeated measure ANOVA was used to explore the interaction between the experimental trial (placebo vs. INDO) and time point (baseline vs. pre-syncope). To further explore any significant interaction effects, paired t tests were then employed. In study 2B, pre and post INDO differences were examined via a paired t test. Based on the sample size (n = 5instudy1B,and n = 4 in study 2B), and the effect size (change in cerebral oxygen) with each intervention (hypocapnia or cerebral hypoperfusion), we calculated that a power of 80% would achieve significance if we observed a mean difference of 19% and 10% in cerebral oxygen with hypocapnia (study 1B) and cerebral hypoperfusion induced by INDO (study 2B), respectively. Given that these changes were observed (and exceeded) in studies 1B and 2B and significance was observed, we feel that despite the small sample size in these studies, the effect size of the intervention was suitable. Results Study 1 effect of hypocapnia on orthostatic tolerance In the isocapnia trial, P ET,CO2 was successfully maintained at baseline levels at pre-syncope (38.3 ± 2.7 mmhg vs ± 2.5 mmhg, respectively; P = 0.; Fig. 2), whereas in the uncontrolled trial P ET,CO2 dropped from 37.2 ± 2.7 mmhg at baseline to 26.3 ± 5.9 mmhg at pre-syncope (P < 0.001; Fig. 2). Tolerance time in the isocapnic trial (572 ± 180 s) was not significantly different from the unclamped trial (544 ± 130 s; P = 0.30; Fig. 1A). Several significant main effects between baseline and pre-syncope were evident, independent of experimental trial, when compared with baseline: reductions in MAP ( 20%), mean MCAv ( 21%), mean PCAv ( 19%), CO ( 61%), and SV ( 24%) and an elevation in HR (+87%) wereevidentatpre-syncope(p 0.003; Table 1 and Fig. 2). Significant interaction (P 0.001) effects between measurement points (baseline vs. pre-syncope) and experimental trial (isocapnia vs. poikilocapnia) were evident; when compared with the poikilocapnic trial, baseline mean PCAv was 2 ± 0cms 1 (+4%; P = 0.03) higher in the isocapnic trial (Table 1 and Fig. 2). At pre-syncope, mean MCAv, MCA CVRi, mean PCAv, PCA CVRi and ventilation were, respectively, 15 ± 4 cm s 1 (+34%; P < 0.001), 0.42 ± 0.25 mmhg cm s 1 (+29%; P < 0.001), 13 ± 6 cm s 1 (+37%; P < 0.001), 0.63 ± 0. mmhg cm s 1 (+26%; P < 0.001) and 18 ± 17lmin 1 (+200%; P < 0.001) higher in the isocapnic trial, when compared to the uncontrolled trial (Table 1 and Fig. 2). Overall, pre-syncopal symptoms scores (out of ) associated with pre-syncope did not differ between the isocapnic trial (26 ± 15) and uncontrolled trials (26 ± 15; P = 0.93). Sex differences. Compared to males, independent of experimental trial, tolerance time was 30% shorter in

6 5208 N. C. S. Lewis and others J Physiol females (628 ± 159 s vs. 487 ± 118 s, respectively; P = 0.001; Fig. 1B). Independent of time point (baseline vs. pre-syncope), and females had a lower SV ( 21.8 ± 9.0 ml; %; P < 0.001), CO ( 1.6 ± 0.3 l min 1 ; 36%; P < 0.001), P ET,CO2 ( 1.8 ± 0.8 mmhg; 5%; P < 0.001), ventilation ( 4.5 ± 3.4lmin 1 ;5%;P = 0.04), and a higher TPRi (+5.6 ± 1.1 mmhg l min 1 ; 29%: P = 0.002), when compared with the males (Table 2 and Fig. 3). Study 1B. P ET,CO2 at rest (normocapnia) was.7 ± 1.8 mmhg and decreased to 22.6 ± 5.0 mmhg following hyperventilation (P = 0.002); these values matched those found at pre-syncope in the poikilocapnic trial of study 1, to within 1 2 mmhg. Compared to normocapnia, gcbf and CDO 2 decreased from ± 5mlmin 1 to 25 ± 5mlmin 1 (relative decrease 29%; P = 0.01) and from 108 ± 14 ml 100 g min 1 to 71 ± 15 ml 100 g min 1 (relative decrease 28%; P = 0.02), respectively, whereas cerebral O 2 extraction increased from 34 ± 4% to 53 ± 10% (relative increase, 56%; P = 0.02) in response to hypocapnia (Fig. 4). In response to hypocapnia, HR increased from 61 ± 7 beats min 1 at rest to 82 ± 7 beats min 1 (P < 0.001), whereas, MCAv and PCAv decreased from 69 ± 10 cm s 1 and 46 ± 3cms 1 at rest to 44 ± 5cms 1 and 28 ± 6cms 1 (P 0.006), respectively (Fig. 4). MAP was unchanged with hypocapnia. Study 2 role of cerebral perfusion on orthostatic tolerance At baseline, significant interaction effects between time and experimental trial (P 0.001) were evident. Baseline mean MCAv was 18 ± 2cms 1 (31%; P < 0.001) lower in the INDO trial, when compared with the placebo trial (Table 3 and Fig. 5). However, OT in the placebo trial (1112 ± 144 s) was not significantly different from the INDO trial (1142 ± 200 s; P = 0.74). Significant main effects for time point were evident, independent of experimentaltrial (placebovs. INDO). For example, when compared with baseline, decreases in MAP ( 23%), mean MCAv ( 28%), CO ( 58%), SV ( 32%) and P ET,CO2 ( 13.5 ± 7.0 mmhg; 33%) were evident at pre-syncope (P 0.003; Table 3 and Fig. 5). At this point, elevations in HR (+113%), TPRi (+86%), and MCVRi (+45%) also occurred. Study 2B. Compared to pre-indo, gcbf and CDO 2 decreased by 37% and 36%, respectively, whereas, cerebral O 2 extraction increased from 34 ± 3% to 57 ± 4% (relative increase, 68%) post-indo (P 0.02; Fig. 6). Compared with pre-indo (68 ± 7 cm s 1 ), MCAv decreased following INDO (46 ± 8cms 1 ; P < 0.001). Heart rate, MAP and P ET,CO2 were unaltered following INDO. Discussion The aim of this study was to independently examine the effect of hyperventilatory-induced hypocapnia, and cerebral hypoperfusion at rest on OT. The main finding from study 1 was that, despite a 35% greater reduction in cerebral perfusion at pre-syncope in the presence of hypocapnia, OT was not impaired. The mechanism explaining this comparable OT is likely to be a compensatory elevation ( 56%) in cerebral oxygen extraction and/or the effect of hypocapnia and cerebral ischaemia acting to maintain blood pressure (Del Pozzi et al. 2014). Two secondary observations were that: (1) ventilation levels preceding pre-syncope in the isocapnic trial was elevated by 76% compared to the uncontrolled trial and; (2) sex differences in OT were apparent, probably due to greater reductions in CO and SV in females rather than CBF regulation. The primary finding of study 2 was that despite a 31% reduction in resting MCAv following A Tolerance Time (S) B Tolerance Time (S) Males Females 0 Unclamped Clamped Condition 0 Unclamped Clamped Condition Figure 1. Mean tolerance time to pre-syncope in study 1 Mean tolerance time to pre-syncope in the unclamped (uncontrolled) and clamped (normocapnic) trials (A), and comparison between sexes (B). Significant main effect for sex; females significantly lower than males (P = 0.001).

7 J Physiol Syncope: role of hypocapnia and cerebral hypoperfusion 5209 Table 1. Absolute cardiorespiratory and cerebrovascular measures at baseline and pre-syncope in the unclamped and clamped trail (study 1) Baseline Pre-syncope Variables Unclamped Clamped Unclamped Clamped Significance Cardiovascular SBP (mmhg) 120 ± ± 8 90 ± ± 14 DBP (mmhg) 71 ± 8 72 ± 6 62 ± ± 11 TPRi (mmhg l min 1 ) 16.7 ± ± ± ± 6.8 HR (beats min 1 ) 62 ± ± ± ± 25 SV (ml) 92.4 ± ± ± ± 13.0 Cerebrovascular SMCAv (cm s 1 ) 98 ± ± ± ± 15,, DMCAv (cm s 1 ) 48 ± 10 ± 11 # 33 ± 9 47 ± 12,, MCA CVRi (mmhg cm s 1 ) 1.33 ± ± ± ± 0.37, SPCAv (cm s 1 ) 79 ± ± 23 # 54 ± 13 ± 17,, DPCAv (cm s 1 ) 39 ± 10 ± ± 6 38 ± 12,, PCA CVRi (mmhg cm s 1 ) 1.86 ± ± ± ± 0.36, Respiratory Breathing frequency (breaths min 1 ) 16 ± 3 14 ± 3 # 23 ± ± 17,, Tidal volume (l min 1 ) 0.82 ± ± 0.21 # 1.15 ± ± 0.51,, Values are mean ± SD. Abbreviations: SBP, systolic blood pressure; DBP, diastolic blood pressure; TPRi, total peripheral resistance index; SV, stroke volume; SMCAv, systolic middle cerebral artery velocity; DMCAv, diastolic middle cerebral artery velocity; MCA CVRi, middle cerebral artery cerebral vascular resistance index; SPCAv, systolic posterior cerebral artery velocity; DPCAv, diastolic posterior cerebral artery velocity; PCA CVRi, posterior cerebral artery cerebral vascular resistance index. Significant main effect for measurement point (baseline differs from pre-syncope in both trails; P < ); significant main effect for experimental condition (unclamped differs from clamped at baseline and pre-syncope; P < ); significant interaction effect (P 0.001): # baseline difference between experimental conditions, pre-syncope differs between experimental conditions (P 0.001). INDO ingestion, OT was unaltered. Secondary to this, thecerebralhypoperfusioninducedatrestviaindo ingestion is associated with a 68% increase in cerebral oxygen extraction. Collectively, cerebral hypoperfusion either at rest (post-indo) or induced by hypocapnia at pre-syncope does not influence OT, findings that are attributable to compensatory increases in cerebral O 2 extraction. Effect of hypocapnia on OT Contrary to our hypothesis, the prevention of hyperventilatory-induced hypocapnia during orthostatic stress did not prolong OT, despite a higher cerebral perfusion at eventual pre-syncope. To our knowledge, only Blaber et al. (2001) have previously investigated the effect of an isocapnic (normocapnic) clamp on OT, and the isocapnic clamp was found to improve OT in four out of five participants, when compared to an uncontrolled condition (poikilocapnic). This improvement was associated with a higher resting MCAv (+18%). In the current study, mean PCAv was 4% higher at rest in the isocapnic trial, yet MCAv was not significantly different, and OT was unaltered. The discrepancy in resting MCAv between the current study and that of Blaber et al. (2001) is perhaps due to the lower sample size, non-steady-state orthostatic stress, and/or methodological issues related to the manual off-line analysis of MCAv (which results in more variability of this measure) employed by Blaber et al. In the isocapnic trial, blood flow velocity in the MCA and PCA at pre-syncope was 34% and 37% higher, respectively, than in the poikilocapnic trial, and this is in agreement with the findings by Blaber et al. (2001) who reported a 28% rise in MCAv at orthostatic intolerance during an isocapnic clamp. It is well known that CBF is highly sensitive to changes in P a,co2, and the development of hypocapnia in the absence of hypotension has previously been associated with the onset of syncope and associated symptoms (Naschitz et al. 1997) induced by cerebral hypoperfusion via cerebral vasoconstriction (Kety & Schmidt, 1946; Immink et al. 2013). For example, a 2% reduction in CBF occurs per mmhg reduction in P a,co2, based on volumetric measures of blood flow in the vertebral and internal carotid arteries (as well as MCAv and PCAv; Willie et al. 2012). Compared to the isocapnic trial, P ET,CO2 at pre-syncope was 12 mmhg lower in the uncontrolled trial; therefore, based on our previous findings (Willie et al. 2012), although regional differences were not apparent, this decline in P ET,CO2 would account for 24% of the decrease in intra-cranial velocity, thus leaving 10 13% of the drop in MCAv and PCAv unaccounted for. Interestingly, in the isocapnic trial, the decline in MCAv and PCAv at pre-syncope was 10% and 8%, respectively; therefore, given the successful clamp of P ET,CO2 in this trial, the decline in CBF appears to be

8 5210 N. C. S. Lewis and others J Physiol attributed to the development of arterial hypotension. It is important to highlight that there is evidence to suggest that the cerebrovascular reactivity to P a,co2 has been shown to be diminished with progressive hypotension (Harper & Glass,1965; Ainslieet al. 2012; Willie et al. 2014), thusthe above estimation of P ET,CO2 contribution to the decline in MCAv and PCAv may be overestimated. Together, these findings indicate that hyperventilatory-induced hypocapnia has the ability to lower the CBF threshold for loss of consciousness by 66%, yet this contribution does not appear sufficient to provoke syncope onset. It appears that the additional decline in CBF ( 33%) is mediated via the development of hypotension, and this appears to be the most important factor in determining cerebral hypoperfusion preceding syncope (see Cerebral autoregulation below). Heightened ventilation during isocapnic lower body negative pressure The associated reductions in MAP with progressive LBNP proffered an expected increase in ventilation. Indeed, unloading the baroreflex (i.e. reducing MAP) potentiates Figure 2. Mean change in cardiorespiratory variables and cerebral blood flow velocity with pre-syncope in study 1 Mean change in mean middle cerebral artery (MCAv) and posterior cerebral artery (PCAv) blood flow velocity, end-tidal carbon dioxide (P ET,CO2 ), minute ventilation, mean arterial blood pressure (MAP) and cardiac output (CO) from baseline to pre-syncope in study 1. Main effect for time, independent of experimental trial; baseline significantly different from pre-syncope (P < ). Main effect for experimental trial, independent of time point; clamped trial significantly different from unclamped trial (P < ). Significant interaction between time point and experimental trial; mean PCAv in the clamped trial significantly different from the unclamped trial at baseline; mean PCAv, mean MCAv and P ET,CO2 in the clamped trial were significantly different from the unclamped trial at pre-syncope (P < ).

9 J Physiol Syncope: role of hypocapnia and cerebral hypoperfusion 5211 Table 2. Absolute cardiorespiratory and cerebrovascular measures at baseline and pre-syncope in males and females (study 1) Baseline Pre-syncope Variables Females Males Females Males Significance Cardiovascular SBP (mmhg) 119 ± ± ± ± 17 DBP (mmhg) 72 ± 7 71 ± 7 63 ± ± 15 TPRi (mmhg l min 1 ) 18.3 ± ± ± ± 3.9 Cerebrovascular SMCAv (cm s 1 ) 100 ± ± ± ± 18 DMCAv (cm s 1 ) 51 ± ± ± ± 14 MCA CVRi (mmhg cm s 1 ) 1.27 ± ± ± ± 0.54 SPCAv (cm s 1 ) 81 ± ± ± ± 20 DPCAv (cm s 1 ) 41 ± ± ± 9 31 ± 13 PCA CVRi (mmhg cm s 1 ) 1.77 ± ± ± ± 0.87 Respiratory Ventilation (l min 1 ) 12.2 ± ± ± ± 19.8 Breathing frequency (breaths min 1 ) 15 ± 3 15 ± 3 23 ± 5 21 ± 9 Tidal volume (l min 1 ) 0.85 ± ± ± ± 0.57 Values are mean ± SD. Abbreviations: SBP, systolic blood pressure; DBP, diastolic blood pressure; TPRi, total peripheral resistance index; SMCAv, systolic middle cerebral artery velocity; DMCAv, diastolic middle cerebral artery velocity; MCA CVRi, middle cerebral artery cerebral vascular resistance index; SPCAv, systolic posterior cerebral artery velocity; DPCAv, diastolic posterior cerebral artery velocity; PCA CVRi, posterior cerebral artery cerebral vascular resistance index. Females significantly difference from males (P 0.04). the chemoreflex and increases ventilation (Heistad et al. 1974). Compared with the poikilocapnia trial in study 1, ventilation at pre-syncope was 18 ± 13lmin 1 (76%; Fig. 2) higher in the isocapnia trial; heighten ventilation during orthostatic stress under isocapnia conditions, or following CO 2 supplementation has also been reported by Blaber et al. (2001) and Howden et al. (2004). Despite trial differences in background P a,co2 and ventilation in study 1, the between-trial decline in MAP was identical (Fig. 2). These findings can be theoretically explained by similar sympathetic nerve activity (SNA), although of different derivation; that is, the reduction in P a,co2 in the poikilocapnic trial would have attenuated the chemoreflex induced SNA (Marshall, 1994). In the isocapnic trial, although the chemoreflex contribution to SNA would have been preserved, the increased ventilation would have elicited a reflex SNA inhibition through pulmonary stretch receptors (Coleridge et al. 1965; Weaver, 1977; Somers et al. 1989). In the end, despite the likely elevated respiratory muscle pump with hyperventilation (Miller et al. 2005) the finding of similar MAPs and tolerance times across isocapnic and poikilocapnic conditions suggests that heightened ventilation did not act to improve orthostatic tolerance. Effect of cerebral perfusion on orthostatic tolerance In study 2, cerebral hypoperfusion was induced through the administration of INDO and, in agreement with previous reports using a range of imaging modalities (Markus et al. 1994; Kastrup et al. 1999; Bruhn et al. 2001; Ivancev et al. 2009), resting MCAv was reduced by more than 30%. In contrast to our hypothesis, however, this reduced CBF reserve at rest did not influence OT. No cardiovascular or respiratory differences between the INDO and placebo trial were evident. The decline in MCAv at pre-syncope was much smaller in the INDO trial (16%) compared with the placebo trial (37%). It is important to highlight that in both trials P ET,CO2 levels were intentionally uncontrolled, and individuals became similarly hypocapnic in both trials; P ET,CO2 declined by 14 mmhg and 13 mmhg in the placebo and INDO trial, respectively. Considering the CO 2 reactivity of the MCAv reported by Willie et al. (2012), the degree of hypocapnia in the current study would have contributed to a 28% decline in the MCAv; thus, the remaining 9% decline in MCAv appears to be attributable to arterial hypotension, a finding highly consistent with study 1. Given that the declineinmapwascomparablebetweentheplaceboand INDO trials, it may be reasonable to suggest that 9% of the pre-syncopal decline in MCAv in the INDO trial was also due to the development of arterial hypotension. It has previously been shown in humans that INDO blunts the cerebrovascular vasoconstrictor response to hypocapnia (Markus et al. 1994; Fan et al. 2010) by %; thus, for the same reduction in P ET,CO2,thedeclinein MCAv would be approximately half of that observed in the placebo trial. Additionally, INDO is a potent and reversible cyclooxygenase 1 and 2 enzyme inhibitor that has been shown to increase cerebral vascular resistance (Wennmalm et al. 1984; Hohimer et al. 1985), thus any additive vasoconstriction evoked by the development of hypocapnia was probably blunted following INDO. Moreover, given that cerebral reactivity to changes in P a,co2 is blunted with arterial hypotension (Harper & Glass, 1965; Willie et al. 2014) the attenuated contribution of hypocapnia

10 5212 N. C. S. Lewis and others J Physiol to cerebral hypoperfusion was an expected finding in the INDO trial. Cerebral autoregulation and orthostatic tolerance Cerebral autoregulation (CA) is the ability of the cerebrovasculature to compensate for changes in perfusion pressure such that CBF is to some extent buffered against change in MAP (reviewed in Willie et al. 2014). We recently reanalysed 41 studies in healthy humans reporting concurrently steady-steady state changes in MAP and CBF for the slope of the % CBF % MAP relationship. In the hypotensive range, the average slope was found to be 0.8 ± 0.8 (Numan et al. 2014). The current study is one of the only studies with a large sample size that examined the effect of hypotension on CBF whilst controlling for the confounding influence of both hypocapnia and pharmacological intervention (e.g. Liu et al. 2013). We 44 MAP (mm Hg) Heart rate (beats min 1 ) Mean MCAv (cm s 1 ) P ET,CO2 (mm Hg) SV (ml) Mean PCAv (cm s 1 ) CO (Lmin 1 ) Females Males Baseline Pre-syncope Time Point 2.0 Baseline Pre-syncope Time Point Figure 3. Sex differences in the mean change in cardiorespiratory variables and cerebral blood flow velocity with pre-syncope in study 1 Mean change in mean middle cerebral artery blood flow velocity (MCAv), end-tidal carbon dioxide (P ET,CO2 ), minute ventilation, mean arterial blood pressure (MAP), heart rate. Stroke volume (SV) and cardiac output (CO) from baseline to pre-syncope in study 1. Females significantly different from males.

11 J Physiol Syncope: role of hypocapnia and cerebral hypoperfusion 5213 observed a 0.4% MCAv decrease per mmhg decrease in MAP, half the slope estimated from the reanalysis of all previous studies (Numan et al. 2014). We speculatethis to be a function of the influence of vasoactive agents used in the majority of previous studies. Oxygen delivery, extraction and OT Following both experiments, on a sub-group of participants, we extended our findings to examine how hypocapnia (severity-matched with study 1 on an individual basis) or cerebral hypoperfusion (prior to and following INDO in study 2) altered cerebral oxygen delivery and extraction. Based on the arterial venous oxygen differences, the findings were highly consistent: despite reductions in oxygen delivery induced by hypocapnia or cerebral hypoperfusion, the brain extracts more oxygen to compensate for the reduction in delivery. We acknowledge that these measures were only made at rest and extrapolation to pre-syncope should be done cautiously. Nevertheless, we sought to examine the effects of changes in CBF in the absence of other circulatory adjustments induced by LBNP. Although common symptoms of syncope were reported in both study 1 and study 2, no participants lost consciousness due to the ethical cut-off just prior to syncope. Once the capacity of the brain to extract oxygen is maximized, further decreases in oxygen delivery must ultimately result in loss of consciousness. An earlier study documented cerebral oxygen extraction increases from 44% at rest to 77% (McHenry et al. 1961) during profound hypotension. On the basis of this study, it was suggested that once CA capacity is maximized, any further reductions in MAP produce a linear decrease in CBF, after which cerebral oxygen extraction must necessarily compensate for all further decreases in cerebral oxygen delivery (Schumann et al. 1998; Derdeyn et al. 2002). Although the absolute increase in cerebral oxygen extraction with hypocapnia (+19%) and cerebral hypoperfusion (+23%) were less than observed by McHenry et al. (1961) (+33%), it is important to note that, unlike the data of McHenry et al., the current data were taken at rest and in the absence of hypotension. Additionally, because of the older hypertensive (MAP = 156 mmhg) population, lack of P ET,CO2 measures, and large BP reduction (75%) at syncope in the study by McHenry et al. (1961)), we must compare the findings cautiously. Nevertheless, the comparable extraction data may indicate that reduction in CBF or gcbf (ml 100g min 1 ) CDO 2 (ml min 1 ) P a,co2 (mm Hg) CO 2 Extraction (%) Normocapnia Hypocapnia MAP (mm Hg) Normocapnia Hypocapnia Figure 4. Mean change in cardiorespiratory variables and cerebral blood flow, cerebral delivery of oxygen and cerebral oxygen extraction at rest with hypocapnia in study 1B Mean ± SD (continuous line) and individual change (dotted lines) in global cerebral blood flow (gcbf), cerebral delivery of oxygen (CDO 2 ), arterial carbon dioxide (P a,co2 ), cerebral oxygen (O 2 ) extraction and mean arterial blood pressure (MAP) from normocapnia to hypocapnia in study 1B. Hypocapnia significantly different from normocapnia (P < 0.001).

12 5214 N. C. S. Lewis and others J Physiol Table 3. Absolute cardiorespiratory and cerebrovascular measures at baseline and pre-syncope in the placebo and indomethacin trails (study 2) Baseline Pre-syncope Variables Placebo Indomethacin Placebo Indomethacin Significance Cardiovascular SBP (mmhg) 133 ± ± ± ± 15 DBP (mmhg) 76 ± 8 74 ± 7 ± 9 64 ± 12 TPRi (mmhg l min 1 ) 10.0 ± ± ± ± 4.7 HR (beats min 1 ) 56 ± 8 51 ± ± ± 23 SV (ml) 67.8 ± ± ± ± 11.1 Cerebrovascular SMCAv (cm s 1 ) 88 ± 9 66 ± 6 # 58 ± 7 53 ± 3,, DMCAv (cm s 1 ) 44 ± 5 27 ± 4 # 27 ± 9 25 ± 5,, MCA CVRi (mmhg cm s 1 ) 1.62 ± ± ± ± 0.30 Respiratory Breathing frequency (breaths min 1 ) 15 ± 4 15 ± 4 17 ± 6 18 ± 6 Tidal volume (l min 1 ) 0.76 ± ± ± ± 0.44 Values are mean ± SD. Abbreviations: SBP, systolic blood pressure; DBP, diastolic blood pressure; TPRi, total peripheral resistance index; HR, heart rate; SV, stroke volume; SMCAv, systolic middle cerebral artery velocity; DMCAv, diastolic middle cerebral artery velocity; MCA CVRi, middle cerebral artery cerebral vascular resistance index; SPCAv, systolic posterior cerebral artery velocity; DPCAv, diastolic posterior cerebral artery velocity; PCA CVRi, posterior cerebral artery cerebral vascular resistance index. Significant main effect for measurement point (baseline differs from pre-syncope in both experiment conditions; P < 0.001). Significant main effect for experimental condition (placebo differs from indomethacin; P < 0.001); significant interaction effect (P 0.001): # baseline difference between experimental conditions (P < 0.001). oxygen delivery, or both, independent of arterial hypotension, is the key factor that elicits the compensatory elevations in cerebral oxygen extraction. It would therefore seem possibly that cerebral oxygen extraction maybe potentially be a deciding factor of OT; as soon as oxygen delivery declines and extraction is maximized, loss of consciousness is imminent. On the other hand, given that systemic perfusion pressure (i.e. MAP) is one of the key determinants of cerebral oxygen delivery, it is important to highlight that despite the varied intervention between trials in studies 1 and 2, the decline in MAP was comparable; therefore, the effectiveness of the baroreflex and its influence upon cerebral oxygen delivery (via maintaining cerebral perfusion pressure) may also be a deciding factor of OT. The multifaceted nature (baroreflex effectiveness to maintain perfusion pressure and oxygen delivery vs. compensatory extraction) of vasovagal syncope is clearly highlighted. Sex-related differences Consistent with previous findings (el-bedawi & Hainsworth, 1994; Franke et al. 2003; Fu et al. 2004; Meendering et al. 2005; Fong et al. 2007), a secondary finding of study 1 was that OT was 30% lower in females compared to males (independent of trial condition i.e. conditions of isocapnia or poikilocapnia). In the current study, both females and males at pre-syncope experienced an average CO drop of 1.4 l; however, compared to males, CO was 32% lower in the females at rest and at pre-syncope. Since HR was comparable between sexes at both time points, this difference was attributed to females having a lower SV ( 37%), supporting the finding of Fu et al. (2004). Despite the reduction in OT in females, we show for the first time the absence of sex differences in CBF-related parameters, indicating similar cerebrovascular regulation at pre-syncope. This finding is not fully corroborated by Wang et al. (2010), who reported a larger reduction in the PCAv ( 24 ± 9%) than in the MCAv ( 18 ± 7%) in females during deg HUT despite very small ( 1%) alterations in blood pressure at this point. The findings by Wang et al. (2010) were interpreted to suggest that women have less efficient autoregulation in the PCA compared to the MCA (Wang et al. 2010). This regional difference was not evident in the current study; however, important between-study differences exist. Namely, individuals in the Wang et al. (2010) study were not all taken to pre-syncope, and BP in the females was 19% lower than males, a difference which was not evident in the current study. Our findings indicate similar CBF regulation at pre-syncope in females. That females reached critical levels of cerebral hypoperfusion sooner than males can be explained by their lower CO reserve. Methodological considerations Until recently it has been commonly reported that changes in transcranial Doppler measurements of the middle cerebral artery is a reliable index of CBF changes as measured by magnetic resonance or ultrasound during