Influence of cerebrovascular function on the ... - Wiley Online Library

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J Physiol 577.1 (2006) pp 319–329

Influence of cerebrovascular function on the hypercapnic ventilatory response in healthy humans Ailiang Xie1 , James B. Skatrud1,4 , Barbara Morgan3 , Bruno Chenuel2 , Rami Khayat1 , Kevin Reichmuth1 , Jenny Lin1 and Jerome A. Dempsey2 Departments of 1 Medicine, 2 Population Health Sciences and 3 Orthopeidcs and Rehabilitation, University of Wisconsin, Madison, WI, USA 4 William S. Middleton Veterans Hospital, Madison, WI 53705, USA

An important determinant of [H+ ] in the environment of the central chemoreceptors is cerebral blood flow. Accordingly we hypothesized that a reduction of brain perfusion or a reduced cerebrovascular reactivity to CO2 would lead to hyperventilation and an increased ventilatory responsiveness to CO2 . We used oral indomethacin to reduce the cerebrovascular reactivity to CO2 and tested the steady-state hypercapnic ventilatory response to CO2 in nine normal awake human subjects under normoxia and hyperoxia (50% O2 ). Ninety minutes after indomethacin ingestion, cerebral blood flow velocity (CBFV) in the middle cerebral artery decreased to 77 ± 5% of the initial value and the average slope of CBFV response to hypercapnia was reduced to 31% of control in normoxia (1.92 versus 0.59 cm−1 s−1 mmHg−1 , P < 0.05) and 37% of control in hyperoxia (1.58 versus 0.59 cm−1 s−1 mmHg−1 , P < 0.05). Concomitantly, indomethacin administration also caused 40–60% increases in the slope of the mean ventilatory response to CO2 in both normoxia (1.27 ± 0.31 versus 1.76 ± 0.37 l min−1 mmHg−1 , P < 0.05) and hyperoxia (1.08 ± 0.22 versus 1.79 ± 0.37 l min−1 mmHg−1 , P < 0.05). These correlative findings are consistent with the conclusion that cerebrovascular responsiveness to CO2 is an important determinant of eupnoeic ventilation and of hypercapnic ventilatory responsiveness in humans, primarily via its effects at the level of the central chemoreceptors. (Received 29 March 2006; accepted after revision 21 August 2006; first published online 24 August 2006) Corresponding author A. Xie: Pulmonary Physiology Laboratory, William S. Middleton Veterans Hospital, 2500 Overlook Terrace, Madison, WI 53705, USA. Email: [email protected]

Changes in cerebral blood flow (CBF) could have an important role in stabilizing the breathing pattern during fluctuating levels of chemical stimuli as seen in sleep apnoea. When cerebral blood flow increases, hydrogen ([H+ ]) is washed away from the central chemoreceptor, reducing the drive to breath and therefore ventilation. Conversely, when cerebral blood flow decreases, [H+ ] increases at the level of the central chemoreceptor, thereby enhancing ventilation. Carbon dioxide (CO2 ), is a powerful ventilatory stimulant and also a potent regulator of the cerebral circulation; furthermore, its ventilatory effects via the central chemoreceptors are moderated by its effect on cerebral circulation. Specifically, hypercapnia causes vasodilatation and increased CBF while hypocapnia causes vasoconstriction and decreased CBF. Previous reports with awake goats using mechanical vascular occlusion techniques to progressively reduce CBF have demonstrated significant modulatory influences on hypercapnic ventilatory responsiveness (Chapman et al. 1979b). We wished to determine whether prevention of the cerebrovascular responsiveness to CO2 had a significant  C 2006 The Authors. Journal compilation  C 2006 The Physiological Society

effect on ventilatory control in humans. If so, it may at least partly explain some of the interindividual differences in ventilatory responsiveness to CO2 measured with steady state methods, which are commonly attributed only to differences in chemoreceptor sensitivity. Furthermore, changes in the cerebrovascular response to CO2 may also help explain some of the enhanced ventilatory responsiveness to transient reductions in Pa,CO2 and the increased propensity for apnoea observed in hypoxic environments (Xie et al. 2001, 2006) and in patients with congestive heart failure (CHF) (Xie et al. 2002, 2005a). To this end we used indomethacin to compromise the cerebro-vascular reactivity to CO2 and thereby determine its effects on ventilatory control. Indomethacin is a potent reversible cyclooxygenase inhibitor, which decreases cerebral blood flow and attenuates the cerebrovascular sensitivity to CO2 (Eriksson et al. 1983; Markus et al. 1994; Bruhn et al. 2001; St Lawrence et al. 2002) without concomitant changes in metabolic rate (Hohimer et al. 1985; Kraaier et al. 1992) or plasma catecholamines (Staessen, 1984; Wennmalm et al. 1984; Green, 1987). DOI: 10.1113/jphysiol.2006.110627

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This unique feature makes indomethacin an ideal tool for investigating the effect of cerebral blood flow on the control of breathing in humans. Methods Subjects

Nine healthy volunteers (5 male, 4 female) with a mean age of 24 (18–35) years and body mass index of 25 ± 2 participated in a pair of experiments with and without indomethacin. Female volunteers were studied in the follicular phase of their menstrual cycles. Participants were non-smokers and free from cardiovascular, pulmonary, and neurological diseases. All subjects received verbal and written instructions outlining the experimental procedure; written informed consent was obtained. All experimental procedures were performed in accordance with the Declaration of Helsinki and were approved by the University of Wisconsin Health Sciences Human Subjects Committee. Measurements

Tidal volume (V T ) and breathing frequency were measured with a pneumotachograph (no. 5719; Hans Rudolph, Kansas City, MO, USA) that was attached to a nasal mask. The mask was checked carefully for leaks. End-tidal oxygen (PET,O2 ) and carbon dioxide (PET,CO2 ) tensions were sampled from the mask and measured using gas analysers (no. S-3A/I and CD-3; Ametek, Pittsburgh, PA, USA). Cerebrovascular regulation was evaluated by transcranial Doppler ultrasonography. A 2 MHz pulsed Doppler ultrasound system (Neurovision 500M, Multigon Industries, Younkers, NY, USA) was used to continuously measure peak cerebral blood flow velocity (CBFV) in the proximal segment of the middle cerebral artery (MCA). Heart rate was obtained from the electrocardiogram, and arterial pressure was measured at 1 min intervals using an automatic arm cuff sphygmomanometer (Dinamap no. 1846SX/P; Critikon, Tampa, FL, USA). Mean arterial pressure (MAP), cerebrovascular resistance (CVR) and the pulsatility index (PI) were calculated using the following formulas: MAP = (1/3 systolic pressure + 2/3 diastolic pressure); CVR ≈ MAP/CBFV; PI = (peak CBFV – end diastolic CBFV)/mean CBFV. All variables were recorded continuously on a computer for off-line analysis using custom-written software.

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right temporal bone window according to the method of Otis & Ringelstein (1996). A nasal mask was attached to a pnemotachograph and then to a T-piece with a gas flow of 40 l min−1 . A three-way valve allowed the addition of O2 , N2 or CO2 to the room airflow. After 10 min of baseline ventilatory measurements, subjects took either 100 mg of indomethacin with 20 ml maalox (Treatment) or 20 ml maalox alone (Control), and then lay in the supine position for 90 min. The ventilatory response measurement was performed immediately after the 90 min of resting study, which was then followed by another 30–60 min of measurements of CBFV, heart rate and blood pressure with room air breathing. The hypercapnic ventilatory response (HCVR) measurement was carried out using a steady state, open-circuit technique (Berkenbosch et al. 1989). Three stepwise PET,CO2 elevations were applied to each subject by adding FI,CO2 at 2%, 4% and 6% each time. The PET,CO2 was elevated within two or three breaths, and then maintained constant for 5 min at each target level. Hypercapnic ventilatory response was tested under normoxia and hyperoxia (FI,O2 = 50%). In the normoxic hypercapnia trials, N2 was added – as necessary – to the breathing circuit to prevent the rise in PET,O2 that resulted from hyperventilation and thereby held PET,O2 constant at the room air level. In the hyperoxic hypercapnia trials, FI,O2 was held constant at 50% throughout the experiment. Each subject underwent two HCVR trials with normoxia and another two with hyperoxia in a sandwich order: one normoxic test → two hyperoxic tests → one normoxic test. The tests were separated by at least 5 min of exposure to room air to allow the circulatory and ventilatory variables to return to baseline levels. The same experiments were conducted on two separate days, with and without indomethacin administration. For female subjects, the two experiments were no longer than three days apart to assure a similar phase of the menstrual cycle. The order of the indomethacin treatment versus control was randomized, with 5 out of 9 subjects receiving the indomethacin first. The CBFV response to a reduction in PET,CO2 was measured during the transition from the end of 6% CO2 inhalation to the beginning of room air breathing in the normoxic HCVR trials. We averaged the CBFV and PET,CO2 for 1 min (19 ± 1 breaths) immediately following the termination of 6% CO2 and then plotted the CBFV against the decrease in PET,CO2 .

Data analysis Experimental setup and protocols

All subjects had the same coffee-free breakfast in our laboratory at 8 a.m. A Doppler probe was fitted at the

During the time course study, while breathing room air, the beat-to-beat resting cardiovascular data were averaged every 5 min and then compared with baseline values and sham values, respectively, using two-way ANOVA  C 2006 The Authors. Journal compilation  C 2006 The Physiological Society

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Table 1. Circulatory variables under each experimental condition Heart rate (beats min−1 ) Normoxia F I,CO2 (%) 0 2 4 6

Cont 62 ± 3 60 ± 2 61 ± 3 71 ± 3

MAP (mmHg)

Hyperoxia

Indo 2∗

57 ± 55 ± 3 56 ± 3 66 ± 4

Cont

Normoxia

Indo

60 ± 3 58 ± 3 60 ± 3 67 ± 3

Cont

58 ± 4 53 ± 3 57 ± 3 62 ± 3

74 ± 3 75 ± 4 75 ± 3 79 ± 3

Hyperoxia

Indo 3∗

78 ± 79 ± 2 79 ± 2 85 ± 2

CVR (mmHg cm−1 s−1 ) Normoxia F I,CO2 (%) 0 2 4 6

Cont

Indo

75 ± 3 75 ± 3 75 ± 4 79 ± 3

81 ± 3 80 ± 2 82 ± 2 88 ± 2

PI

Hyperoxia

Normoxia

Hyperoxia

Cont

Indo

Cont

Indo

Cont

Indo

Cont

Indo

1.8 ± 0.2 1.7 ± 0.2 1.5 ± 0.1 1.2 ± 0.1

2.4 ± 0.4 2.3 ± 0.3 2.3 ± 0.3 2.2 ± 0.3

2.1 ± 0.2 1.7 ± 0.1 1.6 ± 0.1 1.2 ± 0.1

2.7 ± 0.3 2.8 ± 0.4 2.7 ± 0.4 2.3 ± 0.3

1.0 ± 0.2 1.0 ± 0.1 0.9 ± 0.1 0.8 ± 0.1

1.2 ± 0.2 1.1 ± 0.2 1.1 ± 0.2 1.0 ± 0.1

1.2 ± 0.3 1.0 ± 0.1 1.0 ± 0.1 0.8 ± 0.1

1.2 ± 0.2 1.2 ± 0.2 1.2 ± 0.3 1.0 ± 0.1

∗P

< 0.05 compared to control. MAP, mean arterial pressure; CVR, cerebrovascular resistance; PI, pulsatility index; Cont, control group; Indo, indomethacin group.

with repeated measurements. For the HCVR tests, all of the relevant cardio-respiratory parameters were averaged over the last minute at each level of PET,CO2 . The V T , frequency, V E and CBFV were plotted against PET,CO2 , and the slope of each parameter was compared between indomethacin treatment and control under normoxic and hyperoxic conditions by two-way ANOVA with repeated measurements. The slopes of reduction of CBFV against the reductions in PET,CO2 were compared between indomethacin and the controls using Student’s paired t test. The reproducibility of V E /PET,CO2 and CBF/PET,CO2 between repeat trials on the same day was tested during room air breathing under control conditions. Data are reported as means ± s.e.m. Results Effects of indomethacin on air-breathing ventilation and haemodynamics

Indomethacin administration caused a reduction in brain blood flow velocity, which began 30 min after ingestion. By 90 min after ingestion, the CBFV decreased to 77 ± 5% of the initial value, which was significantly lower than the sham value (97 ± 4%, P < 0.05). This reduction in CBFV was associated with a slight increase in both CVR (from 1.8 ± 0.2 to 2.4 ± 0.4 mmHg l−1 cm−1 ) and PI (from 1.0 ± 0.2 to 1.2 ± 0.2) (Table 1). These changes persisted for the entire study duration, including post-HCVR tests. Heart rate went down slightly within 30 min of indomethacin ingestion, and then levelled off at this low level prior to the HCRV tests. By contrast, no change  C 2006 The Authors. Journal compilation  C 2006 The Physiological Society

in heart rate was noted during the sham study, making baseline heart rate significantly lower with indomethacin than with control (57 ± 2 versus 62 ± 3 beats min−1 , P < 0.05) (Table 1). Blood pressure tended to fall slightly over time in the sham study, but no change occurred in the indomethacin study during the 90 min air-breathing period (Fig. 1). As a result, the baseline mean MAP before the HCVR test was slightly but significantly higher in the indomethacin study than in control (78 ± 3 versus 74 ± 3 mmHg, P < 0.05) (Table 1). As shown in Table 2, indomethecin caused a significant hyperventilation in both normoxia and hyperoxia (−2 to −3 mmHg PET,CO2 ), secondary to an increase in ventilation.

Effects of indomethacin on cerebrovascular and ventilatory responses to CO2

The coefficient of variation for the CO2 responses between repeat trials and within subjects was 12 ± 2% for V E /PET,CO2 and 19 ± 5% for CBF/PET,CO2 . During progressive hypercapnia, both CBFV and V E were strongly correlated to PET,CO2 with r 2 values greater than 0.88 in all trials. Indomethacin reduced the mean CBFV reactivity to hypercapnia to less than one-third of its control value, as the response slope was reduced from 1.92 ± 0.17 to 0.59 ± 0.11 cm−1 s−1 mmHg−1 during normoxia (P < 0.05), and from 1.58 ± 0.17 to 0.59 ± 0.09 cm−1 s−1 mmHg−1 during hyperoxia (P < 0.05) (Fig. 2). Correspondingly, indomethacin increased the mean ventilatory response to CO2 by 40%, as the response slope was increased from 1.27 ± 0.3 to 1.76 ± 0.37 l min−1 mmHg−1 (P < 0.05) during normoxia

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and 1.08 ± 0.22 to 1.79 ± 0.37 l min−1 mmHg−1 (P < 0.05) during hyperoxia (Fig. 3). Indomethacin enhanced the hypercapnic ventilatory response mainly by increasing the tidal volume response to PET,CO2 (from 5.8 ± 1.3 to 8.0 ± 1.7 ml mmHg−1 , P < 0.05 during normoxia; from 5.8 ± 1.3 to 7.3 ± 1.4 ml mmHg−1 , P = 0.21 during hyperoxia), with no change in the response slope for breathing frequency. The transition from CO2 inhalation to air breathing was associated with a transient reduction in PET,CO2 , which averaged −14.1 ± 0.4 mmHg in the control trials and −16.9 ± 0.6 mmHg in the indomethacin trials.

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The decrease in CBFV in response to transient reductions in Pa,CO2 was markedly attenuated by indomethacin, as shown in Fig. 4 (1.45 ± 0.15 versus 0.59 ± 0.14 cm−1 s−1 mmHg−1 , P < 0.01). Effect of Pa,O2 on hypercapnic ventilatory response

We tested the hypercapnic ventilatory response under backgrounds of normoxia (PET,O2 = 105−107 mmHg) and hyperoxia (PET,O2 = 383−396 mmHg), respectively. Steady-state hyperoxia reduced the baseline PET,CO2 both with and without indomethacin (Table 2). There was no

Figure 1. Time course of resting CBFV of MCA, heart rate and mean blood pressure A 100 mg oral dose of indomethacin (•) or sham study ( e) at time 0. Cerebral flow velocity was represented as the percentage of the baseline value. Between the first 90 min and the last 35 min, the HCVR interventions were performed. +P < 0.05 compared to baseline; ∗ P < 0.05 compared to sham value at the same time point.  C 2006 The Authors. Journal compilation  C 2006 The Physiological Society

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Table 2. Respiratory variables under each experimental condition PET,CO2 (mmHg) F I,CO2 (%) 0 2 4 6 ∗P

Normoxia Cont 43 ± 1 45 ± 1 49 ± 1 55 ± 1

Hyperoxia

Indo 1∗

41 ± 44 ± 0 48 ± 0 54 ± 0

Cont 39 ± 1† 42 ± 1† 46 ± 1† 54 ± 1

Freq (breaths min−1 )

V T (l) Normoxia

Indo 1∗ †

37 ± 40 ± 1† 45 ± 0† 53 ± 1

Cont 0.4 ± 0.0 0.5 ± 0.1 0.7 ± 0.1 1.1 ± 0.1

Hyperoxia

Indo 0.1∗

0.6 ± 0.7 ± 0.1 1.1 ± 0.2 1.7 ± 0.3

Normoxia

Hyperoxia

Cont

Indo

Cont

Indo

Cont

Indo

0.5 ± 0.1 0.6 ± 0.1 0.8 ± 0.1 1.3 ± 0.2

0.7 ± 0.1 0.8 ± 0.1 1.3 ± 0.2 1.9 ± 0.3

18 ± 1 17 ± 1 19 ± 1 21 ± 1

17 ± 1 17 ± 1 18 ± 1 21 ± 0

18 ± 1 18 ± 1 19 ± 1 21 ± 1

18 ± 1 18 ± 1 20 ± 1 22 ± 1

< 0.05 compared to control. †P < 0.05 compared to normoxia. V T , tidal volume; Freq, breathing frequency.

consistent change in blood pressure or heart rate between the normoxia and hyperoxia trials (Table 1). The mean changes of the cerebrovascular response to CO2 and the ventilatory response to CO2 with indomethacin were not different between the normoxic and hyperoxic trials.

of the ventilatory responsiveness to hypercapnia in the human. This observation provides a reasonable basis for speculation concerning the potential role of the cerebral circulation in the pathogenesis of alterations in the hypercapnic ventilatory response and of periodic breathing in patients with heart failure.

Discussion Our study has demonstrated that indomethecin exaggerates the hypercapnic ventilatory response in association with a significant attenuation of the cerebral vascular reactivity to Pa,CO2 . Thus, these data suggest that cerebral blood flow is an important determinant

Figure 2. Effect of indomethacin on the CBFV responses to PET,CO2 during normoxia (left panel) and hyperoxia (right panel) The upper panel shows individual slopes. The low panel shows group data. Under both normoxic and hypercapnic conditions, indomethacin (•) attenuated the CBFV response compared to the sham study (). There was no difference in the slopes between normoxia and hyperoxia either with or without indomethacin. ∗ P < 0.05 compared to sham.  C 2006 The Authors. Journal compilation  C 2006 The Physiological Society

Methodological considerations

The CBF responsiveness to CO2 was monitored using the technique of transcranial Doppler ultrasound. Its limitations and validation have previously been addressed (Xie et al. 2005a). Even though CBFV is measured in

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only a single brain blood vessel, previous studies have demonstrated that changes in the CBFV in the middle cerebral artery reflect changes in mean cerebral blood flow being measured by a number of other techniques (Bishop et al. 1986; Poulin & Robbins, 1996; Serrador et al. 2000). Very little change in the middle cerebral artery cross-sectional area was noted at the levels of hypercapnia used in this study (Giller et al. 1993; Poulin & Robbins 1996), making CBFV a reliable estimate of the change in the middle cerebral blood flow. The parallel changes in CVR and PI noticed in the present study suggest that the indomethacin-related vasoconstriction and CO2 -induced vasodilatation took place at the distal and small brain vessels (Czosnyka et al. 1996). Likewise, in one subject, we have confirmed that indomethacin (90 min following a 100 mg oral dose) causes similar percentage changes in cerebral blood flow as measured by MRI (69% of baseline) and in CBFV of the MCA as measured by Doppler ultrasonography (68% of baseline) as well as a comparable reduction in cerebrovascular responsiveness to CO2 . Since a similar decrease in blood flow via indomethacin has been observed in the majority of brain regions, including the medullary region (Hohimer et al. 1985; Pourcyrous et al. 1994), the reduction of CBFV should reflect the influence

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of indomethacin on the global as well as medullary CBF, where central chemoreceptors are located. A limitation in the interpretation of our data is that indomethacin affects ventilation not only through its influence on the cerebral circulation but also through the effect of prostaglandins on ventilation. Prostaglandins affect ventilation in different ways according to age. An inhibitory effect has been noted in early life and a stimulatory effect in adult humans (Carlson et al. 1969) and animals (McQueen, 1974). In our adult subjects, we would anticipate that their ventilation and hypercapnic ventilatory responses would, if anything, be depressed when we blocked the stimulating effect of prostaglandins. Accordingly, the observed increase in the hypercapnic ventilatory response – which we attribute to a reduced cerebral vasodilation – may actually be underestimated as a result of indomethacin’s inhibitory influence on prostaglandins. We used a steady-state open circuit method for measuring ventilatory CO2 responsiveness. Accordingly, we maintained a PCO2 gradient between blood and brain. This approach closely mimicked physiological conditions, especially during sleep in which Pa,CO2 would be transiently increased secondary to hypoventilation or apnoea. We

Figure 3. Effect of indomethacin on the ventilatory response to CO2 A, individual slopes; B, group data. Indomethacin (•) increased V E /P ET,CO2 compared to the sham study (). There was no difference between normoxia and hyperoxia in terms of V E /P ET,CO2 . ∗ P < 0.05 compared to sham.  C 2006 The Authors. Journal compilation  C 2006 The Physiological Society

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also used both hyperoxic and normoxic backgrounds during the CO2 response test. Use of a normoxic background ruled out any potential confounding effects of the cerebral vascular constrictions that normally accompany hyperoxia (Leahy et al. 1980; Ellingsen et al. 1987). Our use of hyperoxia suppressed the peripheral chemoreceptor contribution to the ventilatory response to hypercapnia (Lahiri & DeLaney, 1975; Rodman et al. 2001). Furthermore, indomethacin does not have a significant influence on the functional status of the carotid body (Wolsink et al. 1994). Hence the finding that indomethacin also significantly increased the hyperoxic hypercapnic ventilatory response to about the same extent as with the normoxic CO2 response indicates that the increased response was caused primarily by the effect of indomethacin on the environment of the central chemoreceptors. In summary, these lines of correlative evidence strongly support the conclusion that the cerebrovascular chemosensitivity to CO2 has a substantial influence on the ventilatory responsiveness of the central chemorecptors to hypercapnia. One means of further testing this hypothesis would be to use a traditional (Read, 1967) or modified (Duffin et al. 2000) rebreathing type of CO2 response test, during which the PCO2 difference between arterial blood and brain is not influenced appreciably by variations in CBF (Read & Leigh, 1967; Pandit et al. 2003). Accordingly, we would predict that indomethacin would have relatively little effect on the slope of the CO2 rebreathing test for ventilatory responsiveness.

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response to hypercapnia (Kastrup et al. 1999), it follows that the vasoconstrictive function of indomethacin is related to its inhibitory influence on the cyclooxygenase (Therkelsen et al. 1994). On the other hand, the role of prostaglandin-related mechanisms in the inhibitory effect of indomethacin on cerebral vascular CO2 reactivity has been questioned since its effects are only partially reversed by the administration of prostocyclin (Pickard et al. 1980). Moreover, other cyclo-oxygenase inhibitors such as diclofenac, aspirin, naproxen, etc. do not affect cerebral vascular reactivity (Markus et al. 1994; Wagerle & Degiulio, 1994; Pellicer et al. 1999). Alternatively, several other mechanisms for these indomethacin effects have been suggested, such as inhibition of histamine release (Konig et al. 1987), blockade of calcium channels (Northover, 1977), potentiation of the lipoxygenase pathway (Docherty & Wilson, 1987), modifying the extra-cellular pH of vessels (Wang et al. 1993), or through the drug’s radical scavenging effect (Pourcyrous et al. 1993). None of these mechanisms has been conclusively shown to be singularly responsible for the cerebral vascular effects of indomethacin. Modulation of cerebral vascular CO2 reactivity influences ventilatory responsiveness

Although central chemosensitivity is conventionally estimated by determining the slope of the ventilation relative to Pa,CO2 during progressive hypercapnia, central chemoreceptors are not directly stimulated by arterial PCO2 , but rather by [H+ ] in their environment. This stimulus level is more likely better represented by the PCO2 of the venous cerebral outflow or by a mean of arterial

Hemodynamic influence of indomethacin

Indomethacin has been used as a pharmacological model of cerebral ischaemia by other investigators (Hemler et al. 1990; Kraaier et al. 1992). We found that an indomethacin dose of 100 mg attenuated baseline CBF by about 23% of the initial value and the CBFV/CO2 slope to about one-third of its control value in both normoxia and hyperoxia, which is in accordance with previous reports (DeGiulio et al. 1989; Pourcyrous et al. 1994; Kastrup et al. 1999; St Lawrence et al. 2002). The pathway through which indomethacin affects CBF is still obscure. Based on the conflicting findings of indomethaicn on blood perfusion into other systemic organs (Leffler et al. 1986; Stiris et al. 1992), it is still not clear whether the effect of indomethacin is restricted specifically to the cerebrovascular tone or exerts a more general systemic vasoconstriction. Indomethacin is a powerful inhibitor of fatty acid cyclooxygenase, and it thereby blocks the transformation from arachidonic acid to prostaglandin. Since prostaglandin products are able to dilate cerebral vessels and play an important role in the regulation of resting CBF and in the vasodilatory  C 2006 The Authors. Journal compilation  C 2006 The Physiological Society

Figure 4. CBFV response to a reduction in PET,CO2 during the transition from hypercapnia to room air CBFV response to a reduction in P ET,CO2 (from 55.6 ± 1.0 to 41.5 ± 1.0 mmHg in the control trials; 54.6 ± 0.5 to 37.6 ± 0.9 mmHg in the indo trials) during the transition from hypercapnia to room air (n = 8). One subject’s data are missing because he lost his CBFV signal immediately after the end of 6% CO2 inhalation. The other eight subjects consistently showed a reduction in CBFV response to CO2 withdrawal by indomethacin administration compared to the sham study.

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and cerebral venous PCO2 (Fencl, 1986). Besides brain metabolism and CO2 storage, cerebrovascular reactivity is a major determinant of the proportionality between PCO2 changes in arterial blood and brain tissue. Each 10% increase in CBF results in an approximately 0.7 mmHg decrease in brain tissue PCO2 (Fencl, 1986). Arterial hypercapnia increases CBF, and the increased CBF, in turn, will wash CO2 out of brain tissues and narrow the difference between arterial and cerebral venous PCO2 . Thus, in healthy subjects, CO2 inhalation causes an increase in internal jugular venous and brain interstitial fluid PCO2 to a lesser degree than the corresponding increase in systemic arterial blood (Kety & Schmidt, 1948; Fencl et al. 1969), consequently dampening the ventilatory response to CO2 . Indomethacin reduced cerebral blood flow during air breathing and attenuated the increase in cerebral flow velocity during CO2 inhalation; coincidentally a small but significant hyperventilation occurred during air breathing and the ventilatory response slope to hypercapnia increased 40–60% above control. This observation is consistent with the finding of a reciprocal relationship between CBF and diaphragmatic activity in adult goats (Parisi et al. 1988, Parisi et al. 1992). Is the hyperventilation and augmented CO2 response slope solely attributable to an elevated PCO2 (and [H+ ]) stimulus at the level of the medullary chemoreceptors as a result of the reduced CBFV? We addressed this question by estimating the cerebral venous PCO2 based on the cerebral blood flow velocity (Fencl et al. 1969, Fencl, 1986) and found that when V E was plotted versus estimated jugular venous PCO2 (V E /PJV,CO2 ), the 40% increase in the slope of V E /PET,CO2 , which occurred when the CBFV

Figure 5. Ventilatory response to PET,CO2 ( control; e indo) and to jugular venous PCO2 ( control; • indo) Jugular venous P CO2 was estimated using the formula P JV,CO2 = P ET,CO2 + 10.6 − (y − 100) × 0.07, which was derived from the work of Fencl (1969, 1986). The slope of the V E /P ET,CO2 response was increased significantly via indomethacin (also see Fig. 2); however, the slope of the V E /P JV,CO2 was unchanged via indomethacin (P = 0.26).

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responsiveness was reduced via indomathacin, was now eliminated (see Fig. 5). These comparisons serve as illustrations of the significant effects of the cerebral vascular responsiveness to CO2 in determining the gain of the hypercapnoeic ventilatory response in humans. Our data suggest a key role for cerebral vascular responsiveness in ventilatory control in the awake human, which confirms the findings in the awake goat of Chapman et al. (1979a,b), who used a vascular occlusion technique to reduce cerebral blood flow and direct measurements of jugular venous PCO2 to quantify the influence of cerebral flow on the ‘central’ [H+ ] stimulus (also see Introduction). These data also show the important contributions to total ventilatory drive made by the central chemoreceptors in humans. However, they do not rule out the importance of the carotid chemoreceptors in the ventilatory response to CO2 . Indeed, the carotid chemoreceptors are solely responsible for the early phase of the ventilatory response to Pa,CO2 (Smith et al. 2006). In carotid body denervated awake animals, the steady-state hypercapnic response is reduced 40–50% below intact controls (Rodman et al. 2001; Hodges et al. 2005). Moreover, intact carotid chemoreceptors are required for the apnoea that occurs during sleep following a transient ventilatory overshoot (Nakayama et al. 2003). The important role of peripheral chemoreceptor hypersensitivity in destabilizing the breathing control system has also been shown in humans with CHF (Ponikowski et al. 1999) and in healthy humans sleeping in hypoxia (Xie et al. 2006). Clinical implications

Our data provide a reasonable basis for speculating about the role of the cerebral circulation in the pathogenesis of hyperventilation and periodic breathing in patients with CHF. As we previously reported, patients with heart failure and Cheyne-Stokes respiration had an impaired cerebrovascular response to CO2 (Xie et al. 2005a). The magnitude of the reduced cerebrovascular response to CO2 in CHF patients with periodic breathing averaged about 50% less than in normal healthy adults (Przybylowski et al. 2003; Xie et al. 2005a), which is close to the averaged indomethacin effect we observed in healthy subjects. Although intersubject comparisons of Doppler measurements of MCA velocity must be interpreted with cautions, the present findings suggest that the reduced cerebrovascular reactivity will cause an increase in ventilatory responsiveness to CO2 , which is commonly seen during sleep in patients with central sleep apnea (CSA) (Xie et al. 1995; Javaheri, 1999; Solin et al. 2000). With this increased ventilatory chemosensitivity, any transient disturbance to blood gases such as occurs during a sigh, movement arousal, sudden release of upper airway obstruction or termination  C 2006 The Authors. Journal compilation  C 2006 The Physiological Society

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of an apnoea during sleep can initiate or perpetuate periodic breathing because the exaggerated secondary hyperpnoea may drive Pa,CO2 below the apnoeic threshold and cause apnoeas. Furthermore, and consistent with present findings, we have recently demonstrated that reducing the cerebrovascular response to hypocapnia via indomethacin (also see Fig. 4) will also increase the slope of the CO2 response below eupnoea, thereby reducing the CO2 reserve below eupnoea, as seen in patients with CHF and CSA (Xie et al. 2002), and enhancing the propensity for apnoea (Xie et al. 2005b). Finally, a low CBF under baseline air-breathing conditions has been reported in patients with CHF (Rajagopalan et al. 1984; Lee et al. 2001), which might contribute to the chronic hyperventilation found in patients with CHF and CSA due to a chronically elevated CO2 and [H+ ] stimulus in the environment of the central chemoreceptors. The reduction in CBF is certainly not the only mechanism for the increased chemosensitivity or periodic breathing or hyperventilation found in patients with CSA. Previous studies have shown an enhanced ventilatory responsiveness to CO2 in these patients even when the effect of CBF on the response was minimized by using the rebreathing method (Javaheri, 1999; Solin et al. 2000). Indeed, the carotid chemoreceptors are also greatly sensitized in humans with CHF (Solin et al. 2000) and in animal models of CHF (Sun et al. 1999). Furthermore, other potential contributing influences on the enhanced CO2 chemosensitivity above and below eupnoea in CHF include the increased ventilatory drive secondary to raised pulmonary vascular pressures (Solin, 1999; Chenuel et al. 2006) and the destabilizing influence of a decreased cardiac output, and increased circulation time (Batzel et al. 2005). Thus while we believe the present data support the concept of a significant influence of an impaired cerebrovascular CO2 responsiveness to periodic breathing in patients with CHF, we recognize the complexity of ventilatory control in this disease state and emphasize the need for further research to quantify the relative contributions of these various mechanisms to periodic breathing. In summary, the indomethacin-induced reduction in the cerebrovascular response to CO2 was associated with an increase in the ventilatory response to CO2 . This observation raises the possibility that disease states associated with an attenuated cerebrovascular responsiveness to CO2 , such as congestive heart failure, may predispose patients to periodic breathing, in part due to a CBF-mediated augmentation of the ventilatory response to CO2 , both above and below eupnoea. References Batzel JJ, Kappel F & Timischl-Teschl S (2005). A cardiovascular-respiratory control system model including state delay with application to congestive heart failure in humans. J Math Biol 50, 293–335.  C 2006 The Authors. Journal compilation  C 2006 The Physiological Society

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Acknowledgements We are grateful to Dominic Puleo for technical assistance. This study was supported by the VA Research Service, NHLBI and American Lung Association of Wisconsin.