Mechanisms of the cerebrovascular response to ... - Wiley Online Library

Journal of Physiology

J Physiol (2003), 548.1, pp. 323–332 © The Physiological Society 2003

DOI: 10.1113/jphysiol.2002.029678 www.jphysiol.org

Mechanisms of the cerebrovascular response to apnoea in humans Tadeusz Przybyl/owski*, Muhammad-Fuad Bangash†, Kevin Reichmuth†, Barbara J. Morgan‡, James B. Skatrud† and Jerome A. Dempsey* Departments of *Population Health Sciences, †Medicine, and ‡Orthopedics and Rehabilitation, University of Wisconsin-Madison, the John Rankin Laboratory of Pulmonary Medicine, and the Middleton Veterans Administration Hospital, Madison, WI 53705, USA

We measured ventilation, arterial O2 saturation, end-tidal CO2 (PET,CO2), blood pressure (intraarterial catheter or photoelectric plethysmograph), and flow velocity in the middle cerebral artery (CFV) (pulsed Doppler ultrasound) in 17 healthy awake subjects while they performed 20 s breath holds under control conditions and during ganglionic blockade (intravenous trimethaphan, 4.4 ± 1.1 mg min_1 (mean ± S.D.)). Under control conditions, breath holding caused increases in PET,CO2 (7 ± 1 mmHg) and in mean arterial pressure (MAP) (15 ± 2 mmHg). A transient hyperventilation (PET,CO2 _7 ± 1 mmHg vs. baseline) occurred post-apnoea. CFV increased during apnoeas (by 42 ± 3 %) and decreased below baseline (by 20 ± 2 %) during post-apnoea hyperventilation. In the post-apnoea recovery period, CFV returned to baseline in 45 ± 4 s. The post-apnoea decrease in CFV did not occur when hyperventilation was prevented. During ganglionic blockade, which abolished the increase in MAP, apnoea-induced increases in CFV were partially attenuated (by 26 ± 2 %). Increases in PET,CO2 and decreases in oxyhaemoglobin saturation (Sa,O2) (by 2 ± 1 %) during breath holds were identical in the intact and blocked conditions. Ganglionic blockade had no effect on the slope of the CFV response to hypocapnia but it reduced the CFV response to hypercapnia (by 17 ± 5 %). We attribute this effect to abolition of the hypercapnia-induced increase in MAP. Peak increases in CFV during 20 s Mueller manoeuvres (40 ± 3 %) were the same as control breath holds, despite a 15 mmHg initial, transient decrease in MAP. Hyperoxia also had no effect on the apnoea-induced increase in CFV (40 ± 4 %). We conclude that apnoea-induced fluctuations in CFV were caused primarily by increases and decreases in arterial partial pressure of CO2 (Pa,CO2) and that sympathetic nervous system activity was not required for either the initiation or the maintenance of the cerebrovascular response to hyper- and hypocapnia. Increased MAP or other unknown influences of autonomic activation on the cerebral circulation played a smaller but significant role in the apnoea-induced increase in CFV; however, negative intrathoracic pressure and the small amount of oxyhaemoglobin desaturation caused by 20 s apnoea did not affect CFV. (Resubmitted 26 July 2002; accepted after revision 16 January 2003; first published online 14 February 2003) Corresponding author T. Przybyl owski: Katedra i Klinika Chorób Wewn e˛ trznych, Pneumonologii i Alergologii, 02-097 Warszawa ul. Banacha 1A, Poland. Email: [email protected] /

Episodes of obstructive apnoea during sleep and voluntary breath holds during wakefulness in normal subjects perturb the cerebral circulation (Siebler & Nachtman, 1993; Balfors & Franklin, 1994; Hajak et al. 1996). Specifically, there is a progressive rise in cerebral flow velocity during the apnoea followed by an abrupt decrease in the post-apnoea hyperventilation period; however, the underlying mechanisms are unknown. Obstructive apnoeas have complex chemical, mechanical and neural consequences, any of which might alter cerebral blood flow. For example, apnoeas cause hypoxaemia and hypercapnia, both of which are potent vasodilators in the cerebral circulation. In addition, apnoea-induced increases in systemic blood pressure could cause passive increases in

cerebral blood flow that are not counteracted by autoregulation, either because they are too abrupt, or because autoregulatory vasoconstriction is overridden by the vasodilatory effects of hypercapnia and hypoxaemia (EkstromJodal et al. 1972). Negative intrathoracic pressure swings transiently decrease arterial and central venous pressures (Somers et al. 1993); thus, they may reduce cerebral perfusion, especially if they are accompanied by increases in intracranial pressure (Jennum & Borgesen, 1989). Also, apnoeas trigger marked increases in sympathetic vasoconstrictor outflow, mainly via chemoreflex mechanisms. Although the influence of the sympathetic nervous system on cerebral vessels is thought to be minimal, sympathetic activation may alter vascular responsiveness to fluctuations in arterial CO2 tension (Jordan et al. 2000).

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Previous observations of apnoea-induced fluctuations in CFV were made in individuals with obstructive sleep apnoea syndrome, a patient group where co-existing vascular disease (e.g. hypertension and/or atherosclerosis) is prevalent (Shahar et al. 2001). The purpose of the present study was to determine the mechanisms responsible for CFV fluctuations caused by apnoea in the absence of vascular disease. Accordingly, healthy, young subjects performed voluntary apnoeas during wakefulness while we controlled or eliminated, one at a time, potential contributors to the CFV fluctuations.

METHODS Subjects Seventeen subjects (4 women, 13 men) aged 25 ± 7 years (mean ± _2 S.D.) with body mass index of 24 ± 2 kg m (mean ± S.D.) participated in the study. The study requirements were explained in detail to all subjects, and all gave written informed consent prior to participation. The protocol was approved by the University of Wisconsin-Madison Health Sciences Human Subjects Committee and performed according to the Declaration of Helsinki. Participants were not taking any medication, and none had a history of pulmonary, neurological or cardiovascular disease. General procedures All experiments were conducted with subjects supine in a quiet, darkened room with temperature controlled at 20–22 °C. Ventilation (◊ E) was measured through a mouthpiece connected to a pneumotachograph (Model 3700, Hans Rudolph, Kansas City, MO, USA). Oxyhaemoglobin saturation (Sa,O2) was measured continuously with an ear oximeter (Model 3740, Ohmeda, Louisville, CO, USA). Expired air was sampled from the mouthpiece and the end-tidal CO2 tension (PET,CJ) was measured with an infrared gas analyser (Model CD3A, Ametek, Pittsburgh, PA, USA). To estimate intrathoracic pressure, mouth pressure was measured with a transducer (Model MP45-1, Validyne, Northridge, CA, USA) attached to the mouthpiece. Heart rate (HR) was measured from the electrocardiogram. Blood pressure was measured directly using an intra-arterial catheter (Arrow International, Reading, PA, USA) inserted into the radial artery or indirectly by photoelectric plethysmography (Finapres, Ohmeda, Louisville, CO, USA). A 2 MHz pulsed Doppler ultrasound system (Neurovision 500 M, Multigon Industries, Yonkers, NY, USA) was used to measure peak cerebral blood flow velocity (CFV) in the proximal (M1) segment of the middle cerebral artery. The middle cerebral artery was insonated through the right temporal window using search techniques that have been described previously (Otis & Ringelstein, 1996). After obtaining the bestquality signal, the probe was secured using a headband device to provide a fixed angle of insonation. All variables were recorded continuously on paper (Astro-Med K2G, Grass Instruments, West Warwick, RI, USA) and videotape (no. 400A PCM, Vetter, Rebersburg, PA, USA). The signals were also routed to a computer (sampling rate, 120 Hz) for off-line analysis using custom-written software. Experimental protocols Haemodynamic and ventilatory responses to 20 s breath holds were measured in the intact condition and during ganglionic blockade with trimethaphan (n = 8). Responses to steady state hyper- and hypocapnia (see below) were measured under the

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same conditions (n = 9). In these protocols, direct measurements of arterial pressure were made. In a separate group of subjects (n = 7), breath holds were also performed during administration of supplemental oxygen and in some trials, negative intrathoracic pressure accompanied the breath holds (Mueller manoeuvres, see below). In this protocol, arterial pressure was measured indirectly using photoelectric plethysmography. Breath holds (n = 8). During a familiarization visit to the laboratory prior to data collection, subjects practiced 20 s breath holds. They also practiced controlling their breathing patterns in the baseline and post-apnoeic recovery period using auditory and visual feedback of tidal volume, frequency and duty cycle (TI/TTOT). To determine the effects of post-apnoeic hyperventilation and hypocapnia on the cerebrovascular response to breath hold, two breathing patterns were used in the post-apnoeic recovery period. For controlled-mode recovery, subjects returned immediately to the eupnoeic (pre-apnoea) tidal volume, frequency and TI/TTOT. For hyperpnoeic mode recovery, the first three postapnoeic breaths were taken at tidal volume of 2, 3 and 2 times the eupnoeic level, respectively, at 175 % of the eupnoeic frequency, with the eupnoeic TI/TTOT. This breathing pattern was chosen because it mimics the post-apnoeic ventilatory response seen during typical episodes of obstructive sleep apnoea (Morgan et al. 1998). On the day of the experiment, subjects performed 20 s breath holds starting at functional residual capacity. We chose this starting point because we wanted to mimic typical sleep apnoeas. Four to six breath hold trials were performed with each mode of recovery (controlled or hyperpnoeic, order randomized). At least 2 min of eupnoeic breathing were allowed between each trial. In at least two additional trials per subject, the subjects exhaled immediately after the end of breath hold so that an end-tidal sample could be acquired for CO2 analysis. During these trials, which were not included in our assessment of the cardiovascular responses to apnoea, the PET,CJ increased to 7 ± 1 mmHg above the baseline level. Ganglionic blockade. To determine the contribution of autonomic activation and the subsequent blood pressure rise to the cerebrovascular response to breath holds, they were repeated under conditions of ganglionic blockade. Trimethaphan (4.4 ± 1.1 mg min_1, mean ± S.D.) was infused into a forearm vein throughout this part of the study. For each subject the dose was titrated (starting at 3 mg min_1) until the blood pressure response to a 20 s breath hold was abolished. This dose of trimethaphan lowered mean arterial pressure (MAP) by 13 ± 8 mmHg (mean ± S.D.); therefore, we used a concomitant infusion of phenylephrine (10.5 ± 4.8 mg min_1, mean ± S.D.) to return blood pressure to the baseline level. Hypercapnia and hypocapnia (n = 9). Our aim in using ganglionic blockade was to prevent the apnoea-induced rise in blood pressure so that we could determine the effect of this pressor response on CFV. However ganglionic blockade, per se, may affect CFV regulation (Jordan et al. 2000). Accordingly, we measured the effect of ganglionic blockade on CFV responsiveness to alterations in PCO2. Hypercapnia was induced by increasing the inspired fraction of CO2 (FI,CO2) to 2.5 % and 5 % via the breathing circuit. Hypocapnia was produced via voluntary increases in tidal volume to obtain decreases in PET,CJ of 10–13 mmHg. Each trial of hypo- and hypercapnia lasted 6 min. The trials (order randomized) were separated by at least 5 min of room air breathing, which was sufficient time for CFV and PET,CJ to return to baseline levels in all

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subjects. CFV, MAP and PET,CJ during the last 2 min of spontaneous breathing were averaged over all of the baseline periods. The changes in CFV and MAP, elicited by hypo- and hypercapnia, from these baseline values were then calculated. Then, linear regression analysis was used to examine the relationship between CFV and MAP and PET,CJ. This regression analysis was performed separately for the intact and ganglionic blockade conditions. Hyperoxia trials and Mueller manoeuvres (n = 7). In these trials we studied the cerebrovascular effects of: (1) the small amount of oxyhaemoglobin desaturation that occurs during a 20 s breath hold and (2) negative intrathoracic pressure comparable to that produced by obstructive sleep apnoeas. The subjects performed breath holds while breathing room air, during administration of supplemental oxygen (fraction of inspired O2, FI,O2 = 0.50), and during 20 s Mueller manoeuvres in which they attempted to inspire through the mouthpiece against a closed valve. During the Mueller manoeuvres, subjects received visual feedback to assist them in maintaining constant mouth pressure (_40 mmHg). Subjects kept their glottises open for the duration of the manoeuvres; thus, these apnoeas were accompanied by sustained negative intrathoracic pressure (_40 mmHg). All of the trials of this protocol were performed with post-apnoea ◊ E controlled at the baseline level. Data analysis Computations were performed using custom-made software. Mean CFV for each cardiac cycle was determined from the integral of the maximal frequency shift over one cardiac cycle divided by the length of the corresponding cardiac cycle (i.e. velocity–time integral). MAP was calculated as one-third pulse pressure + diastolic pressure. The beat-by-beat values for CFV, MAP and HR were placed into 1 s bins. The peak and nadir values at the termination and recovery periods of each breath hold were computed as 3-beat averages of the actual highest (or lowest) cardiac cycle and the two adjacent cardiac cycles. For each subject, haemodynamic responses to the 4–6 breath hold trials in each condition were averaged, and these average values were used in computation of the group means. Baseline values for intact, trimethaphan alone, and trimethaphan plus phenylephrine conditions were compared using one-way ANOVA with Newman-Keuls post hoc tests. Two-way, repeated measures ANOVAs were used to compare baseline, peak and nadir values for cardiovascular measurements in the intact vs. ganglionic-blocked conditions and in the trials with hyperpnoeic vs. controlled recovery periods. When significant F values for time-by-condition interaction were noted, Newman-Keuls tests were used for post hoc analyses. Time of recovery following an apnoea was defined as the time required for return of the variable to within the 95 % confidence interval around the baseline mean. Linear regression analysis was performed to examine the relationships between PET,CJ and CFV, MAP and ◊ E during steady state hyper- and hypocapnia. The slopes of these linear relationships in the intact and blocked conditions were compared by paired t test. One-way, repeated measures ANOVAs were used to compare peak changes in the cardiovascular measurements during room air and hyperoxic breath holds and Mueller manoeuvres. We used least squares regression analysis of MAP and the CFV:PET,CJ ratio to examine the influence of blood pressure changes evoked by steady state hypercapnia. A value of P < 0.05 was considered statistically significant. Except where otherwise noted, data are expressed as means ± S.E.M.

RESULTS Baseline values Baseline values measured during eupnoeic breathing are shown in Table 1. Trimethaphan caused a significant decrease in MAP that was corrected by concomitant infusion of phenylephrine. HR was significantly increased during trimethaphan infusion, both alone and with phenylephrine.

Response to breath holds Haemodynamic effects of apnoea. In our subjects, 20 s breath holds caused decreases in Sa,O2 (by 2 ± 1 %) and increases in PET,CJ (by 7 ± 1 mmHg) (both P < 0.05). A transient hyperventilation followed resumption of breathing, and PET,CJ was decreased by 7 ± 1 mmHg below baseline (P < 0.05). Breath holds caused biphasic responses in all of the haemodynamic variables we measured (Figs 1 and 2). During the breath holds CFV increased progressively (by 42 ± 3 %, P < 0.05), peaking, on average, 5 ± 1 s after resumption of breathing (Figs 1 and 2). These increases were followed by abrupt decreases in CFV to below the baseline levels (by 20 ± 2 %, P < 0.05), with the nadir values occurring at 18 ± 1 s after resumption of breathing. Blood pressure rose progressively during the apnoeas, reached a peak of 15 ± 2 mmHg (P < 0.05) above baseline at 5 ± 1 s after resumption of breathing, and then fell below baseline (by 5 ± 1 mmHg, P < 0.05) at 24 ± 2 s after the end of the apnoea (Figs 1 and 2). HR increased by 11 ± 2 beats min_1 (P < 0.05), peaking at 4 ± 1 s after the end of the apnoea (not shown). Following this peak, HR returned abruptly to baseline within 10 ± 3 s of the resumption of breathing. Effects of ganglionic blockade on haemodynamic responses to apnoea. During simultaneous infusion of trimethaphan and phenylephrine, apnoea-induced decreases in Sa,O2 (2 ± 1 vs. 2 ± 1 %, P = not significant (NS)) and increases in PET,CJ (7 ± 1 vs. 7 ± 1 mmHg, P = NS) and post-apnoeic decreases in PET,CJ (7 ± 1 vs. _7 ± 1 mmHg, P = NS) were identical to those observed in the intact

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Figure 1. Original record showing typical cardiovascular and ventilatory responses to a 20 s breath hold in the intact condition The PET,CJ of the first post-apnoeic exhalation fails to reflect CO2 accumulation caused by the apnoea because the subject spontaneously inspired upon apnoea termination, thereby diluting the alveolar gas. The values for postapnoeic PET,CJ shown in subsequent figures were determined in separate trials in which subjects exhaled immediately at apnoea termination (see Methods). VT, tidal volume.

condition. Ganglionic blockade virtually abolished the apnoea-induced increase in MAP (1 ± 1 vs. 15 ± 2 mmHg in the intact condition, P < 0.05), but did not affect the post-apnoeic decrease, relative to baseline, in MAP (6 ± 1 vs. 5 ± 1 mmHg, P = NS; Fig. 2). During ganglionic blockade, breath holds increased CFV; however, the increases were smaller than in the intact condition (26 ± 2 vs. 42 ± 3 %, P < 0.05; Fig. 2). In contrast,

the post-apnoeic declines in CFV relative to baseline were similar in the blocked and intact conditions (22 ± 1 vs. 20 ± 2 %, P < 0.05). Baseline HRs were higher during ganglionic blockade, and the HR responses to apnoea were smaller than in the intact condition (2 ± 1 vs. +11 ± 2 beats min_1, P < 0.05; not shown). After resumption of breathing, the peak increases in HR occurred at the same time in the blocked vs. intact conditions (4 ± 1 vs. 4 ± 1 s,

Figure 2. Cerebrovascular responses to 20 s breath holds with hyperpnoeic recovery periods in the intact condition (0) and during ganglionic blockade (9) (n = 8) The dashed vertical lines indicate the duration of the apnoeas and the continuous vertical lines indicate standard errors of the mean (S.E.M.). CFV, cerebral blood flow velocity; MAP, mean arterial pressure. Baseline values for CFV: 65.0 ± 3.6 cm s_1 (intact condition) and 64.0 ± 3.7 cm s_1 (ganglionic blockade). The PET,CJ values noted at apnoea termination were determined in separate trials (see Fig. 1 legend).

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P = NS). In contrast to the prompt recovery of HR in the intact condition, HR during blockade gradually recovered to baseline over the course of 47 ± 5 s.

desaturation (Sa,O2, 99 ± 1 vs. 99 ± 1 %, P = NS). The peak increases in CFV were virtually identical in hyperoxic vs. normoxic conditions (40 ± 4 vs. 43 ± 2 %, P = NS).

Haemodynamic effects of post-apnoea hyperventilation. In the controlled ventilation mode recovery trials, breath holds were terminated with a voluntary return to eupnoeic levels of ◊ E (Fig. 3). This protocol did not affect the decreases in Sa,O2 or increases in PET,CJ that occurred during the breath holds; however, in the post-apnoeic recovery period, PET,CJ, instead of falling abruptly below baseline as it did after breath holds with hyperpnoeic recovery periods, returned gradually to the baseline level. CFV increased to the same extent during breath holds with controlled and hyperpnoeic recovery periods (39 ± 2 vs. 42 ± 3 %, P = NS; Fig. 3). In the controlled recovery period, instead of falling abruptly below baseline as it did during hyperpnoeic recovery, CFV returned gradually to the baseline level.

Hyperoxia attenuated the peak MAP (9 ± 2 vs. 15 ± 4 mmHg, P < 0.05) response to breath hold (Fig. 4), but had no effect on the apnoea-induced increases in HR (6 ± 2 vs. 4 ± 1 beats min_1, P = NS).

The peak values and time of occurrence of the increase in MAP (13 ± 2 vs. 15 ± 2 mmHg at 5 ± 1 vs. 5 ± 1 s, both P = NS) were similar in apnoeas with controlled and hyperpnoeic recovery. Instead of decreasing below baseline as it did during hyperpnoeic recovery, MAP gradually returned to the baseline level. The peak increase in HR was smaller (6 ± 1 vs. 11 ± 2 beats min_1, P < 0.05) and occurred later during controlled vs. hyperpnoeic recovery (9 ± 2 vs. 4 ± 1 s, P < 0.05; not shown). Effects of hyperoxia on haemodynamic responses to apnoea. Administration of supplemental oxygen (FI,O2, 0.50) prevented the apnoea-induced arterial oxygen

Figure 3. Cerebrovascular responses to 20 s breath holds with hyperpnoeic (8) and controlled (ª) recovery periods in the intact condition (n = 8) The dashed vertical lines indicate the duration of the apnoeas and the continuous vertical lines indicate S.E.M. values. Abbreviations as in Fig. 2. Baseline values for CFV: 65.0 ± 3.6 cm s_1 (hyperpnoeic recovery) and 64.9 ± 3.7 cm s_1 (controlled recovery).

Effects of negative intrathoracic pressure on haemodynamic responses to apnoea. Mueller manoeuvres caused a biphasic blood pressure response consisting of a decrease during the inspiratory strain followed by an increase in the post-apnoea recovery period (Fig. 4). Although this time course differed from the progressive increase seen with simple breath holds, the peak increase in blood pressure caused by the two types of apnoea was comparable (10 ± 3 vs. 15 ± 4 mmHg, P = NS). HR, which tended to decrease during simple breath holds, increased during Mueller manoeuvres, with the peak value occurring after resumption of breathing. In spite of these disparate blood pressure and HR responses, the time courses and peak increases in CFV elicited by Mueller manoeuvres and simple breath holds were virtually identical (40 ± 3 vs. 43 ± 2 %, P = NS).

Effects of ganglionic blockade on response to hyperand hypocapnia Hyperventilation produced identical PET,CJ values in the intact condition and during ganglionic blockade (25 ± 1 vs. 25 ± 1 mmHg, P = NS). Elevations in PET,CJ caused by supplementation of inspired CO2 were the same in both

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Figure 4. Cerebrovascular responses to 20 s breath holds performed in normoxia (0), hyperoxia (9), and with sustained negative intrathoracic pressure (Mueller manoeuvres, 3) All breath holds were followed by controlled recovery periods (n = 7). The dashed vertical lines indicate the duration of the apnoeas and the continuous vertical lines indicate standard errors of the mean. Abbreviations as in Fig. 2. Apnoea-induced increases in PET,CJ, measured during normoxic breath holds, were presumed to be the same during Mueller manoeuvres and hyperoxic breath holds. Baseline values for CFV: 34.5 ± 2.4 cm s_1 (normoxic breath holds), 33.8 ± 2.7 cm s_1 (hyperoxic breath holds) and 34.4 ± 2.5 cm s_1 (Mueller manoeuvres).

Figure 5. Relationships between PET,CJ and cerebral blood flow velocity (CFV) and mean arterial pressure (MAP) in the intact condition (0) and during ganglionic blockade (9) (n = 9) Data shown are means ± S.E.M. Asterisks indicate that the slopes of the CFV and MAP responses to hypercapnia were greater in the intact versus the ganglionic blockade condition (P < 0.05).

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conditions (41 ± 1 vs. 41 ± 1 mmHg for 2.5 % FI,CO2 and 48 ± 3 vs. 48 ± 2 mmHg for 5 % FI,CO2, both NS).

DISCUSSION

The slopes of the ventilatory responses to hypercapnia (1.93 ± 0.28 vs. 1.78 ± 0.39 l min_1 mmHg_1) and hypocapnia (_0.75 ± 0.06 vs. _0.80 ± 0.06 l min_1 mmHg_1) were the same in the intact condition and during ganglionic blockade (both P = NS). The slopes of the linear relationships between PET,CJ and CFV (% baseline) during steady state hypercapnia were somewhat steeper in the intact than in the blocked condition (4.70 ± 0.33 vs. 3.88 ± 0.07 % mmHg_1, P = 0.06). This difference was statistically significant when the slopes were calculated using absolute values for CFV (2.93 ± 0.26 vs. 2.49 ± 0.26 cm s_1 mmHg_1, P < 0.05; Fig. 5). The slopes of the MAP responses to steady state hypercapnia were higher in the intact vs. blocked condition (0.77 ± 0.18 vs. _0.17 ± 0.16 mmHg mmHg_1, P < 0.05; Fig. 5). Least squares regression analysis of the CFV: PET,CJ ratio and MAP during hypercapnia in the intact condition revealed that approximately 25 % of the variance in this ratio can be predicted by MAP. In contrast, the slopes of the CFV and MAP responses to steady state hypocapnia were not different in the intact condition vs. ganglionic blockade (1.52 ± 0.11 vs. 1.56 ± 0.17 cm s_1 mmHg_1 and _0.01 ± 0.10 vs. 0.37 ± 0.16 mmHg mmHg_1, both P = NS). The time courses of cerebrovascular responses to hyper- and hypocapnia were comparable in the intact and blocked conditions (Fig. 6).

We have studied the mechanisms responsible for apnoeainduced cerebrovascular changes in healthy, awake humans. CFV increased 42 % above the eupnoeic baseline level during 20 s apnoea and fell 20 % below eupnoea during the transient hyperventilation that followed the apnoea. Our major findings concerning the mechanisms of this response are twofold. First, the prevailing level of PCO2 is the primary regulator of the rise in CFV during and the reduction in CFV following apnoea. Second, the sympathetically mediated blood pressure rise triggered by apnoea plays a smaller, yet still significant, role in producing the apnoea-induced increase in CFV. In contrast, we found no evidence for a direct influence of sympathetic vasoconstrictor outflow on the cerebrovascular responses to apnoea or specifically to changes in PCO2. Likewise, negative intrathoracic pressure and oxyhaemoglobin desaturation played no role in the cerebrovascular response to 20 s apnoea. The following discussion details the assumptions and evidence that underlie these conclusions.

Summary of findings

Critique of methods We used Doppler ultrasonographic measurements of flow velocity to estimate volume of flow through the middle cerebral artery. This method of estimation assumes that the middle cerebral artery diameter remains constant throughout all experimental conditions. We did not

Figure 6. Time courses of cerebrovascular responses to hyper- and hypocapnia in the intact condition (0) and during ganglionic blockade (9) CFV, cerebral blood flow velocity; PET,CJ, end-tidal CO2 tension. All points represent the mean values from 9 subjects.

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measure arterial diameter in this study; therefore, we cannot rule out the possibility that it changed during our interventions, most of which caused substantial perturbations of PCO2 and arterial pressure. Nevertheless, we believe that our velocity measurements are reasonable estimates of flow because previous investigators have shown that the diameter of the middle cerebral artery varies by less than ±4 % during manipulations of arterial pressure (Giller et al. 1993) and PET,CJ (Giller et al. 1993; Valdueza et al. 1997; Serrador et al. 2000) that are even larger than the changes in these variables evoked by our interventions. In addition, velocity and flow through the middle cerebral artery are highly correlated (Bishop et al. 1986; Kirkham et al. 1986). We used photoelectric plethysmography to measure arterial pressure during some apnoeas (the Mueller manoeuvre and hyperoxia trials). Although plethysmographic measurements correlate with intra-arterial measurements during experimental manipulations of arterial pressure (Parati et al. 1989), the absolute values registered by the plethysmograph can sometimes be inaccurate. Nevertheless, we are confident of the accuracy of arterial pressure changes elicited by Mueller manoeuvres and hyperoxic breath holds because they agree both qualitatively and quantitatively with previous measurements made in our lab using direct, intra-arterial methods (Morgan et al. 1993). We estimated steady state CFV and MAP responses to hypocapnia by calculating the slope of the linear relationship between PET,CJ and these variables during voluntary hyperventilation. These estimates must be interpreted with caution, however, because they are based on observations of only one level of hypocapnia. In contrast, our estimates of these responses to increased PCO2 are based on observations made during two levels of hypercapnia produced by CO2 supplementation of the inspirate.

Cerebrovascular response to apnoea: importance of PCO2 In our subjects, nearly two-thirds of the apnoea-induced increase in CFV (i.e. the 26 % increase that remained when blood pressure was held constant vs. the 42 % increase when the blood pressure was allowed to rise) and all of the decrease in CFV during post-apnoea hyperventilation could be attributed to increases and decreases, respectively, in arterial PCO2. The conclusion that PCO2 is the most important determinant of CFV during and after apnoea is supported by several lines of evidence. First, the CFV increase was only partially diminished when the apnoeainduced blood pressure rise was abolished with ganglionic blockade. This diminution cannot be attributed to a decrease in cerebrovascular CO2 sensitivity, because transient and steady state CFV responses to changes in arterial PCO2 were largely unaffected by ganglionic blockade. The diminution in CFV response to hypercapnia that we

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observed during ganglionic blockade (by 17 ± 5 %) can be accounted for by abolition of a covariate, the hypercapniainduced increase in MAP. Second, when post-apnoeic hyperventilation (and the accompanying hypocapnia) were suppressed voluntarily, the post-apnoea reduction in CFV below baseline was abolished. Finally, prevention of the small amount of oxyhaemoglobin desaturation produced by 20 s apnoeas (~2 %) with supplemental oxygen had no effect on the CFV rise. The finding that arterial PCO2 is the primary determinant of CFV during and after apnoea is consistent with current concepts of cerebrovascular regulation. Cerebrovascular smooth muscle is exquisitely sensitive to changes in extracellular PCO2 and H+ concentration; therefore, arterial PCO2 (Pa,CO2) is a powerful regulator of cerebral blood flow. In all mammals studied, hypercapnia and acidosis cause vasodilatation, whereas hypocapnia and alkalosis cause vasoconstriction (Heistad & Kontos, 1983). These responses are qualitatively similar throughout the brain; however, CO2-induced alterations in cerebral blood flow are more pronounced in grey vs. white matter and in cerebellar and brainstem regions vs. cortical areas (Heistad et al. 1976; Busija & Heistad, 1981; Sato et al. 1992; Ramsay et al. 1993). Our estimate of the gain of the cerebral blood flow:Pa,CO2 relationship during hypercapnia (4.7 % mmHg_1) is comparable to that reported by previous investigators (Poulin et al. 1996). Likewise, our estimate of the gain of this relationship during hypocapnia (2.3 % mmHg_1) is comparable to previous reports (Mayberg et al. 1996). In our subjects, post-apnoea decreases in CFV (by 20 ± 2 %) are consistent with what could be predicted on the basis of their steady state responses to hypocapnia and the degree of hypocapnia incurred by the ventilatory overshoot (see Fig. 2). In contrast, the apnoea-induced increases in CFV (42 ± 3 %) are much larger than predicted from the CO2 response alone, an effect we attribute to the apnoeainduced increase in blood pressure.

Effects of apnoea-induced changes in arterial pressure on the cerebral circulation The apnoea-induced increase in CFV was attenuated by more than one-third when arterial pressure was held at baseline levels using ganglionic blockade. The portion of the CVF response thus abolished apparently reflects a passive increase in flow that is secondary to the apnoeainduced pressor response. This effect is somewhat surprising, given the cerebral circulation’s large capacity for autoregulation (Heistad & Kontos, 1983). Our data indicate that autoregulation can be overridden, at least in part, by the relatively rapid changes in arterial pressure caused by apnoea. This finding is consistent with recent evidence that cerebral autoregulation responds more effectively to low- vs. high-frequency changes in blood pressure (Zhang et al. 1998).

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Apnoea and the cerebral circulation

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Effects of negative intrathoracic pressure on the cerebrovascular response to apnoea Aside from transient initial decreases, the CFV changes evoked by Mueller manoeuvres were nearly identical to those caused by simple breath holds. Therefore, negative intrathoracic pressure had no apparent effect on the cerebrovascular response to apnoea even though it produced a rather large decrease in mean arterial pressure (16 mmHg) at the onset of the inspiratory strain (see Fig. 4). We attribute this dissociation of CFV and arterial pressure during the Mueller manoeuvre to the influence of negative intrathoracic pressure on cerebral perfusion pressure (the difference between arterial pressure and cerebral venous pressure, assuming constant intracerebral pressure). The findings of previous investigators suggest that cerebral perfusion pressure may increase during Mueller manoeuvres because of time-dependent differential effects on central venous pressure (an approximation of cerebral venous pressure) and arterial pressure (Somers et al. 1993). On the basis of our data we cannot rule out the possibility that apnoea-induced increases in intracranial pressure, similar to those observed during episodes of obstructive sleep apnoea (Jennum & Borgesen 1989), may negatively influence cerebral perfusion. Nevertheless, it is important to note that, in our experiments, the haemodynamic perturbations associated with negative intrathoracic pressure did not, per se, affect CFV, because the CFV responses were virtually identical during Mueller manoeuvres and simple breath holds. Our findings disagree with those of previous investigators who did not observe progressive increases in CFV during 15 s Mueller manoeuvres or decreases below baseline after resumption of breathing (Reinhard et al. 2000). We attribute the discrepancies in the two studies mainly to differences in apnoea duration. Longer (20 s) apnoeas in our study undoubtedly caused larger amounts of hypercapnia and oxyhaemoglobin desaturation resulting in: (1) greater local vasodilatory effects on cerebral vessels, and (2) more chemoreflex stimulation, which gave rise to a greater post-apnoeic hyperventilatory and blood pressure responses. Also, hypercapnia may have been augmented in the present study because of greater CO2 production during more forceful inspiratory strains (intrathoracic pressure, _40 mmHg vs. _30 mmHg in the previous study). Findings of no post-apnoeic blood pressure overshoot and no change in PET,CJ in the previous study provide support for this explanation (Reinhard et al. 2000).

Effects of sympathetic nervous system activity on cerebrovascular responses to apnoea We found no evidence that basal levels of sympathetic vasoconstrictor outflow limit the amount of apnoeainduced cerebral vasodilatation, because this response was diminished, not enhanced, during ganglionic blockade. Moreover, ganglionic blockade did not affect the gains of

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the transient and steady state CFV responses to hypocapnia. The gain of the steady state CFV response to hypercapnia was reduced by 17 % during ganglionic blockade; however, this effect can be explained by abolition of the hypercapnia-induced pressor response. These data are not consistent with the enhanced hypercapnic vasodilatory response observed after ablation of the superior cervical ganglion in anaesthetized animals (Wei et al. 1980) or with augmented cerebrovascular CO2 responsiveness during ganglionic blockade in conscious humans (Jordan et al. 2000). In the study by Jordan et al. (2000), the slope of the CFV: PET,CJ relationship was approximately 40 % steeper during ganglionic blockade vs. intact preparations, whereas in the present study, the slope was 17 % less steep during ganglionic blockade. We cannot explain this discrepancy between the two studies.

Summary and relevance We found that voluntary breath holds performed by healthy subjects during wakefulness caused cerebrovascular responses qualitatively similar to those reported during episodes of obstructive sleep apnoea, i.e. an oscillatory pattern of progressive rise in cerebral blood flow during the apnoea followed by an abrupt decrease in the post-apnoea hyperpnoeic period. Our experiments demonstrate that this pattern is determined mainly by fluctuations in PCO2. The apnoea-induced pressor response plays a smaller but significant role in causing the apnoeainduced increase in cerebral blood flow, suggesting a failure of cerebrovascular autoregulation. In contrast, negative intrathoracic pressure and mild oxyhaemoglobin desaturation have no effect on cerebral blood flow during apnoea. How generalizable are our findings obtained with voluntary breath holds during wakefulness to clinical sleep-disordered breathing? Our experimental conditions mimicked the hyper- and hypocapnia and the pressor responses caused by typical sleep apnoeas (Shepard 1985; Morgan et al. 1998); therefore, the relative contributions of these perturbations to cerebral blood flow fluctuations should be qualitatively similar in voluntary apnoeas during wakefulness and spontaneous apnoeas during sleep. Furthermore, our findings based on voluntary Mueller manoeuvres strongly suggest that the negative intrathoracic pressure developed during obstructive sleep apnoeas would have little or no further effect on cerebral blood flow beyond the effects of the apnoea, per se. On the other hand, it is important to note that many sleep apnoeas are accompanied by more substantial hypoxaemia than we observed (i.e. Sa,O2 < 90 %), because of a longer duration, lower lung volume and/or reduced initial Sa,O2 (Dempsey et al. 1997). In these circumstances, hypoxaemia may make a significant contribution to the observed increase in cerebral blood flow. In addition, our experimental design did not take into account the effect of sleep, i.e. the known decrease in baseline cerebral blood flow in NREM and

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T. Przybyl/owski and others

increase in baseline flow in REM sleep (Reivich et al. 1968; Sakai et al. 1980). Of even greater importance, our analysis does not consider the effect of an abrupt change in sleep state that accompanies many sleep apnoeas. Transient arousals are accompanied by abrupt increases in cerebral blood flow (Sakai et al. 1980; Hajak et al. 1996) and may have additional interactive influences, along with hypercapnia and blood pressure, on cerebral blood flow changes that accompany apnoeas.

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Acknowledgements The authors are grateful to Mr Dominic Puleo for excellent technical assistance and to Mr Anthony Jacques for expert computer programming. This research was supported by the Veterans Administration Research Service and by National Heart, Lung, and Blood Institute grants to T.P., J.A.D., B.J.M. and a Sleep Academic Award to J.B.S.