Pharmacological FMRI: measuring opioid effects ... - Semantic Scholar

Journal of Cerebral Blood Flow & Metabolism (2007) 27, 414–423 & 2007 ISCBFM All rights reserved 0271-678X/07 $30.00 www.jcbfm.com

Pharmacological FMRI: measuring opioid effects on the BOLD response to hypercapnia Kyle TS Pattinson1,2,3, Richard Rogers1,2, Stephen D Mayhew2, Irene Tracey2,3 and Richard G Wise2,3 1

Nuffield Department of Anaesthetics, Oxford University, John Radcliffe Hospital, Oxford, UK; 2Oxford Centre for Functional Magnetic Resonance Imaging of the Brain, Department of Clinical Neurology, John Radcliffe Hospital, Oxford University, Oxford, UK; 3Department of Physiology, Anatomy and Genetics, Oxford University, Oxford, UK

Opioid binding to the cerebral blood vessels may affect vascular responsiveness and hence confound interpretation of blood oxygen level-dependent (BOLD) responses, which are usually interpreted as neuronal in origin. Opioid binding varies in different brain regions. It is unclear whether opioids alter neurovascular coupling, or whether their effects are purely neuronal. This study used BOLD functional magnetic resonance imaging (FMRI) to investigate the effect of a lopioid agonist remifentanil, on cerebrovascular CO2 reactivity (being one component of neurovascular coupling). Hypercapnic challenges were delivered to human volunteers, while controlling potential opioid-induced respiratory depression. The BOLD signal increase to hypercapnia was compared before and during remifentanil administration. Remifentanil was shown not to have a generalised effect on CO2 responsiveness in the cerebral vasculature. However, it caused a significant reduction in the positive BOLD response to hypercapnia in the bilateral primary sensorimotor cortices, bilateral extrastriate visual areas, left insula, left caudate nucleus, and left inferior temporal gyrus. We conclude that remifentanil does not modulate cerebrovascular CO2 reactivity, as we saw no difference in BOLD response to hypercapnia in areas with high opioid receptor densities. We did however see a focal reduction in areas related to motor control and putative task activation, which we conclude to be related to changes in neuronal activity related to the sedative effects of remifentanil. Our method of controlling CO2 levels effectively mitigated the potential confound of respiratory depression and allowed comparison over a similar range of CO2 levels. We suggest that similar methodology should be used when investigating other potentially vasoactive compounds with FMRI. Journal of Cerebral Blood Flow & Metabolism (2007) 27, 414–423. doi:10.1038/sj.jcbfm.9600347; published online 31 May 2006 Keywords: BOLD; FMRI; hypercapnia; neuro-vascular coupling; opioid; remifentanil

Introduction Functional magnetic resonance imaging (FMRI) is currently being developed as a tool to investigate drug effects in the brain. Pharmacological FMRI studies generally aim to interpret drug-induced changes in stimulus-induced blood oxygen leveldependent (BOLD) response as neuronal in origin. However, a drug may affect the BOLD signal at any point in the pathway from neural activity, to the vascular response that gives rise to a change in the Correspondence: Dr K Pattinson, Nuffield Department of Anaesthetics, Oxford University, John Radcliffe Hospital, Oxford OX3 9DU, UK. E-mail: [email protected] ; published online 31 May 2006 Received 2 February 2006; revised 25 April 2006; accepted 2 May 2006; published online 31 May 2006

BOLD signal. These may be effects on metabolic and synaptic signalling to the vasculature, the properties of the cerebral vasculature, or by changes in systemic cardiovascular or respiratory physiology. These potential effects on the BOLD signal may also be seen in disease states, for example abnormalities in cerebral haemodynamics are seen in patients suffering human immunodeficiency virus (Tracey et al, 1998), stroke, hypertension, and Alzheimer’s disease (Girouard and Iadecola, 2006). The signalling between neurones and the capillaries is generally known as neurovascular coupling, may be metabolic or synaptic, and is considered to cause the increase in capillary blood flow that occurs with neuronal activity. Astrocytes appear to be fundamentally important in this process (Koehler et al, 2006). There are differing theories surrounding the exact mechanisms for this signalling. One theory

Opioid effects on BOLD response to hypercapnia KTS Pattinson et al 415

is that vasoactive neurotransmitters (such as nitric oxide, K + , glutamate, epinephrine) are responsible (Attwell and Iadecola, 2002), alternatively, signalling is mediated through a direct increase in local energy consumption (Magistretti et al, 1999). Drugs may potentially interfere with neurovascular coupling by affecting local transmitter release or metabolic rate. A drug may also have direct effects on the cerebral vasculature. For example, binding to a capillary wall may affect its responsiveness to metabolic signalling or to systemic physiologic changes. Drugs may also change the resting tone of the cerebral vasculature and thereby affect the extent of vasodilatation response to a neural stimulus. Finally, a drug may cause systematic physiologic effects that can interfere with the BOLD response. These include effects on arterial oxygen saturation (SaO2), arterial partial pressure of carbon dioxide (PaCO2) that would be caused by drugs that affect respiration, or by altering the arterial blood pressure. All these potential confounds need to be considered before interpreting BOLD changes as neuronal in origin. A recent review by Wise and Tracey (2006) explores the prospects and difficulties of pharmacological FMRI in greater depth. The aim of this study is to investigate the regional effects of an opioid (remifentanil) on cerebrovascular CO2 reactivity, while controlling for the respiratory effects of the drug. Hypercapnic challenges effectively act as a model for the ‘vascular properties’ part of the BOLD response pathway. We did not aim to investigate either the specific effects on neuronal activity or on neurovascular coupling (i.e., signalling). In common with many centrally acting compounds remifentanil is known to modify cerebral blood flow (Wagner et al, 2001) and has profound effects on respiration. It therefore serves as a good candidate on which to test our proposed methodology for measuring pharmacologically induced changes in cerebrovascular responsiveness. Remifentanil is a short-acting opioid analgesic that is used in anaesthesia. Its ultra-short contextinsensitive half-life (3 to 4 mins) allows rapid adjustment of plasma levels when infused using a computer-controlled pump pre-programmed with a pharmacokinetic model of the drug (target-controlled infusion). We chose remifentanil because it is a good model for opioids that are in current clinical use, but it is more convenient to use in an experimental setting. The short half-life means that it can be administered by infusion and plasma levels can be changed rapidly. The drug and no-drug scans can therefore be acquired during the same session, minimizing inter-session differences. In terms of volunteer safety, it is possible to achieve a relatively strong opioid effect with remifentanil, but if there are any signs of adverse drug reaction, the infusion can be terminated with the confidence that the drug will wear off within minutes. In addition to its analgesic properties, and in common with all other opioids, remifentanil de-

presses breathing. Respiratory depression causes an increased arterial partial pressure of CO2 (PaCO2), which then increases the baseline BOLD signal throughout the brain. Unless this CO2 effect is accounted for, then it may be a significant confounding factor in the interpretation of FMRI studies (Cohen et al, 2002). Hypercapnia is an elevated PaCO2. It provides a cerebrovascular challenge, allowing us to map vascular responsiveness using FMRI (Rostrup et al, 2000). Hypercapnia induces a substantial increase in cerebral blood flow without a change in oxygen metabolism (Kim and Ugurbil, 1997; Vorstrup et al, 1984), and hence induces an increase in BOLD signal. Changes in cerebral vascular responsiveness to CO2 have been used to investigate disease progression, for example after carotid endarterectomy (Vesely et al, 2001). Investigation of a drug’s effect on cerebral vascular responsiveness using hypercapnic challenges was proposed as an important method for pharmacological FMRI studies (Stein, 2001). Such a technique would help an investigator understand more fully how a drug acts in the central nervous system. A recent positron emission tomography (PET) opioidergic radioligand study (Baumgartner et al, 2006) mapped the distribution of opioid receptors in the brain. The study showed high opioid binding in the insular cortex, thalamus, and the anterior cingulate cortex, and lower densities of opioid receptors in the primary and secondary somatosensory cortex. If remifentanil were to have an effect on CO2 responsiveness mediated through the opioid receptors then we would expect to see a pattern of changes in the BOLD response to hypercapnia that reflects the distribution of opioid receptors. While opioids induce hypercapnia through respiratory depression, this paper describes a methodology that controls for this, allowing us to examine the BOLD reactivity to our externally administered CO2 challenges over a normal range of CO2 values. Using CO2 as a probe of vascular reactivity has two main benefits over eliciting a neuronal response (e.g., visual stimulation), firstly the input stimulus, the end-tidal partial pressure of CO2 (PETCO2) (which correlates closely with PaCO2 in healthy subjects) is easily measured and secondly the global changes produced allow mapping of CO2 responsiveness throughout the whole brain. The method we present is aimed at understanding the possible vascular influences of opioids that may affect our interpretation of pharmacological FMRI studies and could be extended more generally to human pharmacological FMRI studies and to studying the vascular effects of disease.

Materials and methods The study was approved by the Oxfordshire Clinical Research Ethics Committee and volunteers gave written, Journal of Cerebral Blood Flow & Metabolism (2007) 27, 414–423


informed consent. Nine healthy volunteers (1 female, mean age7s.d., 3277.2 years) were examined on two different occasions. They were excluded if they were taking medication or drugs acting on the central nervous or respiratory systems for therapeutic or recreational use. A medical history and, where appropriate, a physical examination were performed to ensure that the subjects were fit and well (American Society of Anesthesiologists physical status 1) and that there was no contraindication to remifentanil or to magnetic resonance imaging. The first examination was a medical screen to ensure that volunteers tolerated remifentanil, determined by an anaesthetist monitoring blood pressure and oxygen saturation. The second occasion was FMRI combined with respiratory challenges and a remifentanil infusion to an estimated plasma concentration of 1.0 ng/ml. Volunteers fasted before all visits (6 h for solids and 2 h for clear fluids) and were supervised for 1 h after termination of the infusion.

Breathing System A breathing system was constructed that had minimal resistance to breathing, allowed rapid changes in inspired gas concentration, and eliminated rebreathing of expired gases. The resistance to breathing was kept minimal by using wide-bore respiratory tubing as a reservoir for inspiration. The individual gases were delivered separately to a mixing chamber near the subject, which facilitated rapid changes in gas concentrations. The total fresh gas flow rate was 30 L/min, which eliminated rebreathing of expired gases. The subjects wore a tight fitting facemask (Hans Rudolph, KA, USA), which was attached to the breathing system. To counteract the respiratory depression of remifentanil, subjects were instructed to remain awake and adjust their PETCO2 levels by varying their breathing according to a target PETCO2 value displayed on a screen. The target values were determined before the experiment by measuring PETCO2 values at rest and after 2 mins of a CO2 challenge. During the experiment the subjects were instructed to maintain these levels. This method was designed in order to counteract the rise in PETCO2 from remifentanil-induced respiratory depression. During scanning, phasic hypercapnia was induced by delivering an inspired concentration of 5% CO2 alternating with 0% CO2, each for a period of 2 mins. The carrier gas was a mixture of O2 and air, the composition of which was manually adjusted to maintain a mildly hyperoxic endtidal partial pressure of O2 (PETO2) at 187 mm Hg to minimise the effect of changing arterial oxygen levels on the BOLD signal. The experimental protocol lasted 36 mins.

tained with a pump (Graseby 3500 TCI incorporating ‘Diprifusor’, SIMS Graseby Ltd, UK) (Gray and Kenny, 1998) that was pre-programmed according to a pharmacokinetic model of remifentanil (Minto et al, 1997a, b). The infusion was started 16 mins after the FMRI scanning commenced, and was terminated at the end of the experiment. We chose this concentration of remifentanil because in pilot studies we found that higher concentrations caused subjects to fall asleep and therefore not comply with the protocol. Oxygen saturations and heart rate were monitored continuously and non-invasive blood pressure was recorded every 2 mins (In Vivo Research, Orlando, FL, USA). PETCO2 and PETO2 were determined using rapidly responding gas analyzers (model CD-3A & S-3A; AEI Technologies, Pittsburgh, PA, USA) and continuously displayed and recorded with a data acquisition device (PowerLab 8, AD instruments, Colorado Springs, CO, USA) connected to a laptop computer using dedicated software (Chart 5, AD instruments, Colorado Springs, CO, USA). During the last 10 secs of each CO2 challenge a prompt appeared on the screen for the subject to rate their breathlessness. A breathlessness scale from 0 to 10 (0 for no breathlessness to 10 for the worst breathlessness ever experienced) was reported by pressing a button the appropriate number of times. These cardiovascular and respiratory data sets were compared using paired t-tests. A P-value of < 0.05 was considered significant.

Functional Brain Imaging Imaging was performed at 3 T with a Siemens Trio system using an eight-channel head coil. Whole-brain gradient echo-planar imaging was performed giving T2* weighting or BOLD contrast with a voxel size of 3 3 3 mm3 and an echo time of 30 ms. Each volume comprised 50 contiguous axial slices, 3 mm thick. Volumes were acquired continuously with a repetition time of 3 secs. For each subject, a T1-weighted structural scan (208 contiguous 1 mm axial slices with 1 1 1 mm3 voxels) was acquired. This was used for anatomic overlay of brain activation and to assist in placing individual subject data into a common stereotactic space. The responsiveness of BOLD signal to hypercapnia (defined as the BOLD signal change per unit change in PETCO2) was evaluated over the brain and compared between no-drug (the first 16 mins of the experiment) and remifentanil (the final 16 mins of the experiment) periods. Four minutes were given for the remifentanil to reach target concentration, and the data from this period were not included in the comparison.

Drug Infusion

Image Analysis

A target-controlled infusion of remifentanil (at a solution concentration of 10 mg/ml) was delivered via an indwelling intravenous cannula inserted into a vein in the left forearm. A plasma concentration of 1.0 ng/ml was main-

Analysis was performed using FEAT (FMRI Expert Analysis Tool) Version 5.55, part of FSL (FMRIB’s Software Library, www.fmrib.ox.ac.uk/fsl). The following pre-statistics processing was applied; motion correction

Journal of Cerebral Blood Flow & Metabolism (2007) 27, 414–423


using MCFLIRT (Jenkinson et al, 2002); non-brain removal using BET (Smith, 2002); spatial smoothing using a Gaussian kernel of full width at half maximum 5 mm; mean-based intensity normalisation of all volumes by the same factor; high-pass temporal filtering (Gaussianweighted laser speckle flowmetry straight line fitting, with sigma = 180.0 secs). Time-series statistical analysis was performed using FILM with local autocorrelation correction (Woolrich et al, 2001). Voxel-wise statistical analysis was extended to a second (group) level in a mixed effects analysis using FLAME. Z (Gaussianised T/F) statistic images were thresholded using clusters determined by Z > 2.3 and a (corrected) cluster significance threshold of P = 0.05 (Worsley et al, 1992). Registration to high resolution and standard images was performed using FLIRT. To provide a fair comparison between no-drug and remifentanil periods we therefore excluded from the analysis those volumes acquired when the PETCO2 was greater than the maximum level achieved with the CO2 challenge in the no-drug period. This was performed by modelling such periods as a regressor of no interest in the linear model of the time series data. Six motion parameters (translations and rotations around three axes) were included as regressors of no interest in the model to account for motion-related signal variance that was not corrected by the initial motion correction procedure. A region of interest analysis was performed to identify drug-induced changes in BOLD response to CO2 during the no-drug and remifentanil periods in areas with high opioid receptor densities (Baumgartner et al, 2006) (anterior cingulate cortex, insular cortex, and thalamus) and areas with lower densities of opioid receptors (primary and secondary somatosensory cortices). Predefined masks were created that corresponded with anatomic areas. The CO2-related signal changes in the no-drug and remifentanil states in those regions were compared using one-tailed paired t-tests with a P-value of 0.05 as the threshold for significance.

PETCO2 during the experiment, which largely negated the rise in PETCO2 usually seen with opioidinduced respiratory depression (Babenco et al, 2000). The group average PETCO2 over time is plotted in Figure 1. In both baseline and hypercapnic conditions remifentanil administration was associated with a small but significant rise in PETCO2, averaging a 1 mm Hg increase for both baseline breathing and hypercapnic challenges for the remifentanil condition compared with no-drug infusion. Breathlessness ratings were obtained four times during no-drug and during remifentanil periods. Median scores were 1/44 (0 to 2) for the ND period, and 0/44 (no score above 0) for the remifentanil period. Arterial oxygen saturations remained at 98% to 99% throughout the experimental protocol in all subjects. In no subject was there any episode of desaturation.

Table 1 Cardiovascular and respiratory parameters (n = 9) mean (7s.d.) No drug Systolic blood pressureNS Diastolic blood pressure** Mean blood pressure* Heart rate# End-tidal O2 (during baseline condition)NS End-tidal O2 (during hypercapnic challenge)NS End-tidal CO2 (during baseline condition)** End-tidal CO2 (during hypercapnic challenge)**

120 70 103 60 189

(10) (10) (10) (5) (3)

Remifentanil 119 67 101 57 187

(9) (9) (8) (5) (3)

187 (3)

187 (3)

40 (1)

41 (2)

47 (1)

48 (2)

NS, no significant difference. *P < 0.05, **P < 0.01, #P < 0.001.

Results Cardiovascular and Respiratory Data

The physiologic data is summarised in Table 1. Blood pressure during the no-drug and remifentanil infusion periods was similar. There was no significant change in the systolic pressure, whereas the small reduction in diastolic pressure was statistically significant, as was the small reduction in mean blood pressure. There was a small, significant reduction in heart rate. By manually adjusting the inspired oxygen concentrations we managed to achieve good control of PETO2 during the experiment. There was no significant difference between PETO2 during the nodrug and remifentanil conditions either during baseline breathing or during the hypercapnic challenges. By instructing the subjects to control their breathing we also managed to achieve adequate control of

Figure 1 Plot of PETCO2 averaged over nine subjects for the duration of the experiment. Remifentanil infusion was commenced at 16 mins and is shown in the box. Journal of Cerebral Blood Flow & Metabolism (2007) 27, 414–423


Functional Magnetic Resonance Imaging

As previously reported (Rostrup et al, 1994) there was a significant global BOLD signal increase in response to hypercapnia. This was generally larger in grey matter than white matter. The left side of Figure 2 shows the BOLD response to hypercapnia in two representative axial slices, the upper picture (A) during the no-drug state, and the lower picture (B) with remifentanil infusion. It is difficult to see differences between these images because the effect of a CO2 challenge on the BOLD response is so strong and any remifentanil-induced modulation is small. The right-hand side of Figure 2 (C) therefore shows the difference between the no-drug and remifentanil conditions (i.e., paired group subtraction of A and B). This reveals areas with a significantly smaller increase in the BOLD response to hypercapnia with remifentanil in the left and

right sensorimotor cortices, left and right extrastriate visual areas, left insula, left caudate nucleus, and the left inferior temporal cortex. The coordinates and Z-scores are shown in Table 2. In none of the regions of interest was remifentanil shown to significantly modulate the BOLD response to hypercapnia. The results are summarised in Table 3. Figure 3 graphically shows the relationship between PETCO2 and the BOLD response for the anterior cingulate cortex in one subject, and is representative of all the regions of interest for each of the subjects examined. This shows that the PETCO2 range for the no-drug and remifentanil periods is similar, and the relationship between PETCO2 and BOLD is similar between the no-drug and remifentanil periods. Using the data from the region of interest analysis, we examined the power of the study. On average

Figure 2 BOLD response to hypercapnic challenge, in nine subjects. The images consist of a colour-rendered statistical map of changes in activation (Z-scores) registered onto high-resolution structural scans that have been transformed to standardized brain geometry (Talairach space). The standard threshold for voxel activation was taken as Z > 2.3 (P < 0.05). Color is used to display the Z-scores for voxel activation, from red (lowest Z-score) to yellow (highest Z-score) in (A and B) and for reduced voxel activation from dark blue (lowest Z-score) to bright blue (highest Z-score) in (C). Anatomic left (L) and right (R) are marked with labels indicating relevant anatomic sites. z coordinates are distance above ( + ) or below ( ) the intercommisurial plane in mm. (A) BOLD response to hypercapnia without drug infusion. (B) BOLD response to hypercapnia during remifentanil infusion. (C) Brain areas showing reduced BOLD response to hypercapnic challenge with remifentanil infusion as compared with the no-drug state. (Subtraction of B from A).

Table 2 Coordinates of local maxima of significant reductions in the BOLD response to hypercapnic challenges with remifentanil, values derived from cluster-based analysis Left Region

x

y

z

Sensorimotor Inferior temporal Extrastriate visual areas Insula Caudate

40 34 44 38 18

16 12 64 20 28

58 42 10 8 2

Right Maximum Z-score

x

y

z

maximum Z-score

3.74 3.8 3.4 3.67 3.44

44 — 28 — —

20 — 70 — —

42 — 4 — —

3.91 — 3.48 — —

The most significant maximum is listed for each anatomic location. Coordinates are in mm in standard space of MNI. x, distance right (+) or left ( ) of the mid saggital line; y, distance anterior (+) or posterior ( ) from a vertical plane through the anterior commisure; z, distance above (+) or below ( ) the intercommisurial plane.


Opioid effects on BOLD response to hypercapnia KTS Pattinson et al

Table 3 Mean (s.d.) changes in BOLD signal (arbitrary units) with hypercapnic challenges from region of interest (ROI) analysis,

419

derived from three-dimensional masks based upon anatomic regions drawn in standard space, for no drug and remifentanil infusion (Remi) periods in the anterior cingulate cortex (ACC), left (L) and right (R) insular cortex (Ins), thalamus (Thal), primary somatosensory cortex (SI) and secondary somatosensory cortex (SII) ROI’s with high opioid binding

No drug Remi P-value

ROI’s with low opioid binding

ACC

Ins (L)

Ins (R)

Thal (L)

Thal (R)

SI (L)

SI (R)

SII (L)

SII (R)

217 (40) 212 (57) 0.34

180 (34) 174 (45) 0.27

170 (28) 168 (48) 0.41

149 (34) 144 (47) 0.26

151 (31) 141 (40) 0.13

164 (37) 159 (46) 0.27

253 (105) 236 (97) 0.16

164 (37) 158 (42) 0.16

157 (29) 152 (38) 0.27

P-value from two-tailed t-test.

extrastriate visual areas, left insula, left caudate nucleus, and the left inferior temporal gyrus. The physiologic data revealed a small decrease in blood pressure, no change in PETO2, and a small increase in PETCO2 with remifentanil.

Cardiovascular and Respiratory Data

Figure 3 Plot of PETCO2 against BOLD signal in the anterior cingulate cortex (derived from the region of interest mask) for one subject. The no-drug and remifentanil infusion periods are plotted separately.

there was 80% power to detect 12.4% drug-induced modulation in the amplitude of the BOLD response to hypercapnia, at a significance level of P < 0.05. For example, if normally there was a 2% BOLD signal increase in response to a hypercapnic challenge, we would be able to detect as significantly reduced, a drug-induced modified BOLD response to hypercapnia of 1.75%.

Discussion The most important finding of this study was that we found no generalised modulation of the BOLD response to hypercapnia with remifentanil. There was a large global increase in BOLD signal in response to hypercapnia for both the no-drug and remifentanil conditions. A region of interest analysis based on pre-defined masks showed no modulation of the BOLD response to hypercapnia in predefined areas with both high and low opioid binding across the whole brain volume. However, clusterbased analysis revealed significantly smaller positive in the BOLD responses to hypercapnia in the left and right sensorimotor cortices, left and right

The small reduction in blood pressure during remifentanil administration is unlikely to be a confounding factor. A study in anaesthetised rats (Zaharchuk et al, 1999) showed that gradual blood pressure decreases that were within the range of effective cerebral autoregulation (mean arterial pressure of 50 to 140 mm Hg) were not associated with significant changes in cerebral blood flow or the BOLD signal. Larger sudden changes in blood pressure have been shown to affect the BOLD response (Kalisch et al, 2001) but the changes we saw were well within autoregulatory limits and were small (2% decrease in mean blood pressure) and therefore are unlikely to cause confounds in our FMRI data sets. Similarly the small reduction in heart rate is unlikely to be a relevant confound. By adjusting inspired O2 concentrations we have maintained PETO2 at approximately 187 mm Hg throughout the experiment. The small changes seen are physiologically insignificant. Larger changes in arterial O2 pressure may affect the BOLD signal, especially if a subject’s arterial O2 saturations are allowed to fall below the normal range (hypoxia causes desaturation of haemoglobin and increased cerebral blood flow) (Gupta et al, 1997; Rostrup et al, 2005) or if they are administered much higher O2 levels (Kennan et al, 2004; Sicard and Duong, 2005). Alterations in PaCO2 have profound effects on cerebral blood flow and therefore perturbations in PaCO2 can potentially modify the BOLD response. Traditionally, the relationship between PaCO2 and cerebral blood flow has been considered to be linear (Grubb et al, 1974). Recent studies have challenged these assumptions. Ide et al (2003), found an exponential relationship between PETCO2 and cerebral blood flow measured with transcranial Doppler ultrasound. This has implications for the interpretaJournal of Cerebral Blood Flow & Metabolism (2007) 27, 414–423


tion of BOLD signal changes at varying PETCO2 levels. The effect of hypercapnia on the stimulus-evoked BOLD signal has been studied with conflicting results. Corfield et al (2001) found no modulation of the BOLD response to visual stimulation with hypercapnia up to 47 mm Hg, these results were confirmed in a study by Hoge et al (1999). However, a study by Cohen et al (2002) found that hypercapnia caused a linear reduction in the BOLD response to visual stimulation, and a study by Bandettini and Wong (1997) reported attenuation of BOLD contrast motor activation-related signal changes during hypercapnia. Posse et al (2001) reported an enhanced BOLD response to visual stimulation with mild hypercapnia (PETCO2 up to 50 mm Hg) but saturation of the stimulus-evoked BOLD response when PETCO2 levels exceeded 70 mm Hg. The differing conclusions of these studies may reflect differences in experimental design, particularly differences in nature and duration of the stimuli. However, it would be fair to conclude that BOLD FMRI is sensitive to global changes in cerebral blood flow and that changes in PETCO2 (even in the physiologic range) may affect FMRI contrast, and may not be easily accounted for. An example of systemic pharmacological confound is illustrated in a study in rats that investigated the effect of anaesthesia on cerebrovascular reactivity (Brevard et al, 2003). This study failed to consider that the BOLD response to a hypercapnic challenge might be affected by an increased baseline PaCO2 caused by anaesthesia-induced respiratory depression. The resting PaCO2 levels rose from 38 mm Hg in the non-anaesthetised state to 47 mm Hg with anaesthesia. The two conditions (awake and anaesthetised) were compared over different, nonoverlapping CO2 ranges. Since the relationship between BOLD and PaCO2 is unlikely to be linear over the extended range of CO2 levels in Brevard et al’s study (38 mm Hg conscious baseline to 62 mm Hg anaesthetised with 5% CO2 challenge), it is impossible to say that the reduced reactivity is a direct neuronal effect of anaesthesia, rather than an indirect effect related to the effect of hypercapnia on the BOLD response. These studies emphasise the importance of maintaining the same PaCO2 levels for the baseline and CO2 challenge if using BOLD to investigate pharmacological effects on cerebrovascular reactivity using drugs that may in addition affect respiration and thus CO2 levels. In our study we have trained subjects to control their own breathing. Therefore, the baseline PETCO2 and the raised PETCO2 during the hypercapnic stimulus are almost the same during both no-drug and remifentanil conditions. We have therefore examined the drugs effect on CO2 reactivity on the same portion of the CO2–BOLD response curve thereby mitigating confounds of changes in baseline CO2 that would be caused by remifentanil. Journal of Cerebral Blood Flow & Metabolism (2007) 27, 414–423

We recorded breathlessness ratings because hypercapnia is reported to cause dyspnea with associated specific neural activation (particularly in the anterior insula) (Banzett et al, 2000; Evans et al, 2002). To avoid breathlessness we raised the PETCO2 by considerably lower amounts than in studies that were investigating breathlessness. A few subjects in our study reported mild breathlessness with hypercapnia during the no-drug condition, no one reported breathlessness during the remifentanil infusion. Opioids are known to inhibit breathlessness, so this is not surprising. We saw few changes in brain areas known to mediate breathlessness and can therefore assume that the effect was weak.

Functional Magnetic Resonance Imaging

The aim of this study was to assess the influence of remifentanil on the BOLD response to CO2 and to examine any regional differences that may be related to the distribution of opioid receptors. Although Baumgartner’s study (Baumgartner et al, 2006) identified regional distribution of opioid receptors with PET, the technique was unable to determine whether this binding took place on receptors on neurones, capillaries, astrocytes, or on other cells. The study did not specifically determine effects of opioids on the local vasculature. There is a body of evidence to suggest that opioids may be important in regulation of cerebral blood flow (Benyo and Wahl, 1996). Opioid receptors have been identified on the bovine cerebral capillary endothelium (Peroutka et al, 1980) and a study in rats suggested that they may have their effect by mediating nitric oxide release (Stefano et al, 1995). Another study in rats suggests that endogenous opioids may be important in the regulation of cerebral blood flow during hypercapnia (Dora et al, 1992). Hamel et al (1988) demonstrated differing reactivity to various vasoactive substances in a selection of isolated feline cerebral arteries. There appears to be marked variation between species and whether investigating endogenous or exogenous opioids. Furthermore, most animal studies are performed either under anaesthesia, or using in vitro techniques, and therefore may not be directly applicable to intact human beings. Clinical studies in humans suggest that there is no marked generalised effect of remifentanil on global cerebral CO2 reactivity (Klimscha et al, 2003) when measured with transcranial Doppler ultrasound. High but not moderate dose remifentanil was shown to reduce cerebral blood flow velocity, possibly because of reduced cerebral metabolic rate (Paris et al, 1998). A contrast-enhanced MRI study of remifentanil showed greater increases in cerebral blood flow and volume in areas known to have lower densities of opioid receptors (Lorenz et al, 2000). Unfortunately, there is a paucity of human data regarding regional differences in cerebrovascular


CO2 reactivity or capillary binding of opioids. If there are regional differences in cerebral vascular opioid receptor distribution with important effects on vascular tone and neurovascular coupling, then we may expect to see regional variations in vascular responsiveness to CO2. We therefore hypothesized that brain areas previously identified with pain processing that contain the highest opioid receptor densities would be most likely to have their vascular responsiveness modified by remifentanil. Opioids may also affect the signalling between neurones and the cerebral capillaries (neurovascular coupling). As discussed above, vasoactive neurotransmitters have been implicated as mediators of this intracellular signalling (Attwell and Iadecola, 2002). Stefano et al (1995) implicate nitric oxide as an important mediator of some of the cerebrovascular effects of opioids. Drug effects on this metabolic signalling may be responsible for an unexpected change in the BOLD signal. Our study did not aim to look at the effects of remifentanil on neurovascular coupling, because there is currently no simple method of isolating the signalling process from the neuronal and vascular response. Our methodology only intended to examine changes in cerebrovascular CO2 reactivity, which may be caused by drug effects on the cerebral vessels. We did not find any generalised effect of remifentanil on the BOLD response to CO2, nor did there appear to be significant changes in BOLD response in regions known to be involved with pain processing other than a small area of reduced activation in the posterior insula. Our results appear to disagree with those of Lorenz et al (2000) who showed increased cerebral blood flow and volume with remifentanil that appeared to be most pronounced in areas less rich in opioid receptors. However, their study differs from ours in two important aspects: Firstly, they were looking at changes in baseline haemodynamics (cerebral blood flow, cerebral blood volume, and mean transit time) whereas we were examining the vascular response (above baseline) to the CO2 stimulus. This means that our study could not reveal agonist effect beyond those on cerebral vascular reactivity. To demonstrate an agonist effect with FMRI one approach would be to map regional changes in baseline cerebral blood flow using an arterial spin-labelling technique. Secondly, the region of interest analysis in Lorenz et al’s study was based on larger anatomic areas in the brain (frontal grey, occipital grey, parietal grey, striatal grey, thalamic grey, and white matter) whereas we chose our region of interest analysis based on specific opioid receptor distributions revealed by Baumgartner et al (2006). It appears therefore that the lack of changes in BOLD signal as a function of receptor density means that any drug–receptor binding does not have a great effect on vascular reactivity. This may be because the vascular or metabolic effect of binding may not be strong

enough to cause significant vascular effects, or the effect may be smaller than our study could detect. We found focal reductions in the BOLD response to CO2 with remifentanil unrelated to the distribution of opioid receptors. We interpret these as changes in BOLD as changes in neuronal activation induced by remifentanil that are related to either the input function (watching the screen with the desired and actual PETCO2 value), cognitive processing of this information, or the desired task (motor output to breathing) and are caused by the mild sedative and respiratory depressive effects of the drug. Although one may expect to see reduction in neuronal activity in other brain regions with remifentanil, our experimental paradigm will only reveal changes that are related to it. Similarly, Heinke and Schwarzbauer (2001) investigated the effect of subanaesthetic isoflurane on a visual search task and showed changes in a specific network of brain regions that were related to this task. The cluster analysis revealed several areas of a decrease in BOLD response to hypercapnia with remifentanil infusion. These areas are primarily involved with putative task activation (motor cortex and caudate), and sensory processing (sensory cortex and extrastriate visual areas), and may reflect a mild sedative effect of remifentanil. There are two potential explanations for the observed reduction in BOLD response to hypercapnia seen in the sensorimotor areas. Firstly, the respiratory depressive effect of remifentanil means that subjects are not maintaining respiration as effectively as without drug. This is supported by slightly higher PETCO2 levels during the remifentanil. Simply put, the subject is putting less effort into the task. The second explanation is that the respiratory depressive effects of remifentanil mean that respiration is under greater conscious control during the remifentanil infusion. Therefore, there is increased neural activation and an increase in the baseline BOLD signal. Since greater conscious effort would be required during the baseline (no CO2 challenge) to maintain PETCO2 levels to (almost) normal, there may be a saturation of the BOLD response and a smaller relative increase in BOLD than during the no-drug condition. Neural correlates of volitional respiration have been successfully recorded in the sensorimotor areas both with FMRI (Evans et al, 1999; McKay et al, 2003) and with electroencephalography (Davenport et al, 1986). The changes seen in our study fit with observations in the above studies. We interpret the changes seen in the extrastriate visual areas to be related to attention to the visual information presented during a task and its relation to motor output (Astafiev et al, 2004) and similar in cause as discussed above for the sensorimotor areas, that is either caused by less effort being put into the task or a greater conscious control causing a raised baseline BOLD signal with saturation of the BOLD response. A small area of the left posterior insula also Journal of Cerebral Blood Flow & Metabolism (2007) 27, 414–423


demonstrated reduced activation, which may reflect less breathlessness (although the effect was mild in the no-drug state) or modulation of sensory processing by remifentanil. Overall, our interpretations of these changes are that remifentanil is causing a sedative effect and therefore reduced neuronal activation in a brain matrix that corresponds to task activation related to volitional control of breathing. There was also reduced activation in the inferior temporal gyrus, which is an area particularly susceptible to imaging artefacts related to changes in volume of respiratory passages. It is important to note, however, that our experimental paradigm was designed to control CO2 levels rather than to examine changes in neuronal activity. In conclusion, the most important finding of this study was that remifentanil does not appear to have a generalised effect on cerebrovascular CO2 reactivity, and there appears to be no modulation of CO2 reactivity in a pattern relating to opioid receptor density. By comparing drug and no-drug conditions over the same CO2 range we have eliminated potential confounds of differing BOLD responsiveness with differing baseline CO2 levels. We believe the specific changes we have seen in BOLD response to hypercapnia are because of the effect of remifentanil on respiratory control and its mild sedative effect. The finding that remifentanil does not have a direct effect on BOLD response to CO2 is beneficial for interpreting pharmacological FMRI with opioids. It means that changes seen in the BOLD response to a neuronal stimulus can be interpreted with more confidence as they are unlikely to be confounded by a drug effect on the cerebral vasculature. This is particularly important for drugs that are known to have profound physiologic effects, such as other opioids, sedatives, and anaesthetics. We have described a method of examining a drug effect on cerebrovascular CO2 reactivity, which we believe is an essential step in future pharmacological FMRI studies.

Acknowledgements We acknowledge the generous support of the Association of Anaesthetists of Great Britain and Ireland, The UK Medical Research Council (KP and RW) and The Wellcome Trust (RW, Grant code 067037). We thank Dr Jaideep Pandit for his invaluable assistance with this study.

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