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Effects of cocaine on blood flow and oxygen metabolism in the rat brain: implications for phMRI
Magnetic Resonance Imaging 25 (2007) 795 – 800
Effects of cocaine on blood flow and oxygen metabolism in the rat brain: implications for phMRI Laura Ceolin, Adam J. Schwarz, Alessandro Gozzi, Torsten Reese, Angelo Bifone4 Department of Neuroimaging, Psychiatry CEDD, GlaxoSmithKline Medicines Research Centre, 37135 Verona, Italy Accepted 11 January 2007
Abstract The effects of cocaine on cerebral blood flow and tissue oxygen levels in the rat brain were investigated with concurrent laser Doppler flowmetry and fluorescence quenching spectroscopy. Responses elicited by mild hypercapnia were used as calibration to assess the effects of cocaine on oxidative metabolism. Intravenous cocaine challenge of 0.5 mg/kg induced significant increases in tissular oxygenation and perfusion in all regions investigated (primary motor cortex, medial prefrontal cortex and dorsal striatum). Mild hypercapnia, a challenge that affects haemodynamics but not metabolism, elicited comparable changes in blood flow but substantially larger changes in tissue oxygen levels. These differences in tissue oxygen build-up suggest that increased oxidative metabolism is a significant component of the cerebral metabolic response to acute cocaine challenge. The implications for the interpretation of pharmacological MRI data are discussed. D 2007 Elsevier Inc. All rights reserved. Keywords: Cocaine; Functional MRI; fMRI; phMRI; Brain metabolism; Oxygen consumption; LDF; CBF
1. Introduction Functional magnetic resonance imaging (fMRI) methods have been applied to map the central haemodynamic response to cocaine challenge as a surrogate for changes in brain activity in humans [1] and laboratory animals [2–5]. This fMRI approach, sometimes referred to as bpharmacological MRIQ (phMRI) [6–9] is promising for the investigation of the central activity of cocaine and other psychoactive drugs at a system level. However, the complex effects of cocaine on neuronal activity, brain metabolism and vascular response are not fully understood, and a link between haemodynamic changes and the underlying neuronal activity remains to be demonstrated. One of the primary actions of cocaine in the brain is blockade of dopamine transporters, resulting in an increase in synaptic dopamine levels. These modulations in the mesolimbic dopamine pathway have been associated with the psychostimulant effects and reinforcing properties of cocaine [10]. Conceivably, changes in neuronal activity induced by cocaine might induce changes in brain metabolism, which in turn might trigger a central haemodynamic 4 Corresponding author. Tel.: +39 045 921 9707. E-mail address: [email protected] (A. Bifone). 0730-725X/$ – see front matter D 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.mri.2006.10.022
response. Indeed, acute cocaine challenge in the rat induces a widespread central haemodynamic response whose amplitude and temporal profile vary with anatomical location [2,3]. However, other mechanisms have been invoked, including changes in blood pressure [11,12], direct vasoactivity of dopamine [7,13] and innervation of blood vessels by dopaminergic neurons [13]. Recently, we have investigated the correlation between dopamine levels and relative cerebral blood volume (rCBV) changes in the rat brain by concurrent microdialysis and fMRI [3]. Interestingly, an fMRI response was observed also in brain structures, like the motor cortex, where cocaine did not elicit detectable changes in dopamine levels, thus indicating that in these areas the haemodynamic response is not directly driven by local dopamine modulations. Here we have investigated the effects of cocaine on brain tissue oxygen levels and their correlation with haemodynamic changes elicited by the drug. Specifically, we have applied fluorescence quenching spectroscopy and laser Doppler flowmetry (LDF) to simultaneously measure brain tissue oxygen tension (pO2) and cerebral blood flow following acute cocaine challenge in the anaesthetised rat. Changes in tissue pO2 reflect transient imbalance between oxygen consumption and supply, and are sensitive to local changes in oxygen metabolic rate [14]. Three different brain
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regions, the primary motor cortex (M1), medial prefrontal cortex (mPFC) and dorsal striatum (dStr) were investigated. These regions were chosen because each shows a different temporal rCBV response profile [2] and a different relationship with local changes in dopamine levels as detected by microdialysis [3]. For comparison, we have also measured the response to CO2 challenge in the primary motor cortex and in dStr. Mild hypercapnia has been shown to induce a haemodynamic response without significantly affecting brain metabolism [15] and has been proposed as a calibration to measure changes in oxygen metabolic rates [16–18]. The aim of the present study was twofold: (a)
to measure the temporal dynamics of tissue oxygen levels and blood flow following cocaine challenge; (b) to investigate the correlation between haemodynamic response elicited by cocaine and the underlying changes in oxygen metabolism. 2. Materials and methods 2.1. The Oxylite system A combined Oxylite/LDF system (Oxford Optronix, Oxford, UK) was used to simultaneously measure tissue oxygen tension and blood flow in the rat brain. Oxylite technology uses a ruthenium luminophor located at the tip of an optical fiber, which conveys light pulses generated by a diode to the photoactive material. The lifetime of the resultant fluorescence is inversely proportional to pO2 at the probe tip. The diameter of the optical fiber is 220 Am. The Oxylite probe is bundled with another optical fiber (200 Am diameter) for the laser Doppler flow measurement. The tips of the probes are closely positioned to provide simultaneous measurements of the two parameters over approximately the same volume. All probes were precalibrated by the manufacturer, and the calibration parameters were scanned into the system prior to each experiment. Oxylite presents several advantages over polarographic measurements of pO2 for the scope of this experiment. Firstly, Oxylite does not consume oxygen during the measurement and allows continuous monitoring of oxygen levels for extended periods of time [19]. Moreover, the Oxylite probe measures an average pO2 over a sensing volume that is substantially larger than that probed by microelectrodes [20]. Thus, Oxylite is less sensitive to the heterogeneity of oxygen concentration on the length scale of capillaries. 2.2. Animal preparation All experiments were carried out in strict accordance with Italian animal welfare legislation and GSK internal ethical review. Male Sprague-Dawley rats (Charles River, Italy), weighing 250–350 g, were anesthetized with 3% halothane in a 30%/70% O2/N2 mixture using a facemask. The animals were tracheotomised and artificially ventilated with a
mechanical respirator (Inspira, Oxford, UK). Upon tracheotomy and throughout surgery, the anaesthetic level was maintained at 1.5–2.0%. Before each incision, the surgical area was treated subcutaneously with 0.3 ml marcaine, a topical anesthetic. The left femoral artery and vein were cannulated with a PE50 polyethylene catheter for the infusion of compounds and for monitoring of arterial blood pressure. Subsequently, the animals were placed in a stereotaxic frame for the insertion of the combined dual fluorescence-quenching/laser Doppler flow probe. The animals were divided into three groups, with the combined Oxylite/LDF probe located in either the primary motor cortex, dStr or medial prefrontal cortex. A hole was drilled through the parietal bone, and the probe was inserted perpendicular to the brain surface according to the coordinates from dura mater [28] as follows: motor cortex (M1) AP +2.2 mm, ML +2.8 mm, DV 2.5 mm; dStr AP +1.0 mm, ML +2.5 mm, DV 4.1 mm; mPFC AP +2.7 mm, ML +0.5 mm, DV 3.5 mm. After surgery, the halothane concentration was decreased to a maintenance level of 1%. d-Tubocurarine (0.25 mg/kg h iv, Sigma-Aldrich, Milan, Italy), dissolved in saline heparin (25 UI/ml), was infused through the cannulated artery to maintain muscle relaxation. Mean arterial blood pressure and heart rate were monitored continually throughout the experiment with a blood pressure transducer (Biopac Systems Corp., Goweta, USA) connected to the arterial catheter. The body temperature was monitored with a rectal probe and maintained at 37.5F18C using a heating pad. The volume of ventilation was adjusted to maintain blood pCO2 within physiological range and blood pO2 N 100. A period of stabilisation of 2 h was allowed before starting the pO2/LDF measurement. Blood samples of ca. 200 Al were collected from the arterial catheter before and immediately after the end of pO2/LDF data acquisition, and blood gas levels measured using a blood gas analyzer (AVL, Roswell, GA, USA). The protocol of the present study, including anaesthetic regimen, animal preparation, monitoring and stereotaxic coordinates, was very similar to that we used in a previously published phMRI/microdialysis study [3] in order to facilitate comparison of cocaine-induced pO2 and LDF changes with the rCBV response. Three groups of animals were infused intravenously over 1 min with either a cocaine challenge (Sigma, 0.5 mg/kg in saline, 1.0 ml/rat) or vehicle (saline, 1.0 ml/kg). Group sizes were as follows: M1, n = 7 (5 cocaine, 2 saline); dStr, n = 10 (4 cocaine, 6 saline); mPFC, n = 11 (6 cocaine, 5 saline). A dose of 0.5 mg/kg of cocaine was chosen for consistency with previous phMRI [2,3], microdialysis [3] and deoxyglucose autoradiography studies [21]. Two separate groups of animals (M1 and dStr, n = 4 each) were challenged with CO2 through the mechanical ventilation system. In order to determine the concentration of CO2 that best mimicked the LDF response to cocaine, different amounts of CO2 (in the range 0.05 to 0.1 l per minute, corresponding to 2.5–5% CO2) were added to the O2/N2
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Fig. 1. Temporal profiles of LDF, pO2 and MBP changes following cocaine and vehicle (saline) challenge measured in (A) the motor cortex (M1), (B) the dorsal striatum and (C) the mPFC.
mixture for periods of 10 min, followed by 10 min of recovery. Basal blood pO2 and pCO2 levels were in the range 180–210 and 34–37 mm Hg, respectively, for all groups. No significant changes in blood gas levels were observed after cocaine challenge. In the subjects challenged with CO2, no significant changes in pO2 were observed, while pCO2 levels rose slightly, but did not exceed 41 mm Hg, well within the range of mild hypercapnia conditions. Tissue pO2 and LDF values were measured for 10 min before and 30 min after vehicle, cocaine or CO2 challenge. At the end of the experiment, the animals were euthanized with an overdose of anaesthetic followed by cervical dislocation. 2.3. Data analysis Time courses for MBP, pO2 and LDF were exported from the Biopac software at a time resolution of ~10 s per data point. Data were further rebinned to 1-min intervals for the time-course figures for clarity of display. In order to compare changes in tissular pO2 for similar changes in local LDF between cocaine- and hypercapnia-challenged animals, each time course was normalised to the mean of a 10-min baseline period prior to the challenge and thus expressed as a fractional change, denoted yLDF and ypO2, respectively. For each animal, the average yLDF and ypO2 over a 3-min time window at the peak response was used as a summary
statistic of the relative change in each parameter. This captured stable LDF and pO2 values corresponding to a steady-state regime. Statistical analysis was performed on the fractional changes. For the hypercapnia-challenged animals, the CO2 challenge (0.05 or 0.1 l per minute) that yielded a yLDF closest to the mean yLDF in the corresponding cocaine group was used, so that the LDF changes matched as closely as possible those arising following cocaine challenge. The values of yLDF and ypO2 were compared between cocaineand hypercapnia-challenged groups in the striatum and motor cortex by two-sample T-test (Statistica v. 6.1, StatSoft Inc.). Threshold for statistical significance was considered as P =.05. Results are quoted and shown as meanFS.E.M. unless otherwise indicated.
3. Results 3.1. Temporal profiles The temporal profiles of LDF, pO2 and mean blood pressure (MBP) changes following acute cocaine challenge are shown in Fig. 1. In the motor cortex, both LDF and pO2 showed a rapid increase to a peak about 4 min postinjection and returned to baseline values within ~15 min. In the striatum, LDF and pO2 responses were more sustained, with the tail of the response more variable between subjects, as
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Fig. 2. Temporal profiles of LDF, pO2 and MBP changes following hypercapnia challenge measured in (A) the motor cortex (M1) and (B) the dorsal striatum.
reflected by the larger error bars at late time points. In both brain regions, the time courses of LDF and pO2 changes were positively correlated (mean intra-animal cross-correlation: q =0.66). In the mPFC, LDF changes were more sustained than for pO2, which rose to a broad maximum after 5–6 min, before returning more slowly toward baseline values after ~30 min. Vehicle injection did not produce significant LDF or pO2 changes, and neither cocaine nor saline significantly affected blood pressure. The temporal profiles of LDF, pO2 and MBP following CO2 challenge are shown in Fig. 2. In the motor cortex, LDF rose rapidly, reached its peak value within 2–3 min and remained sustained over the duration of the experiment. Tissue oxygen tension rose more slowly and reached a plateau after 6–7 min from the beginning of the CO2 challenge. In the dStr, LDF peaked after 4 min and slowly decreased towards baseline, while pO2 reached a plateau. Transient negative changes in arterial blood pressure were observed following CO2 challenge, but they did not reach statistical significance.
4. Discussion and conclusion The temporal profiles of LDF and pO2 following cocaine challenge were positively correlated in all the regions examined in this study. Interestingly, the LDF and pO2 time courses varied with anatomical location, with a faster response in the motor cortex and more sustained responses in the mPFC and in the dStr. This observation is consistent with previous rCBV measurements [2,3]. The rCBV responses reported in Ref. [3], in particular, were acquired under very similar experimental conditions and animal preparation procedures to those used in the present
3.2. Amplitude changes in LDF and pO2 Cocaine challenge elicited peak fractional LDF changes of 0.24F0.08 and 0.29F0.16 in M1 and dStr, respectively. These were accompanied by increases in tissue pO2 of similar magnitude, 0.28F0.03 and 0.29F0.13, respectively (Fig. 3). Hypercapnia challenge yielded LDF changes of 0.30F0.14 and 0.20F0.05 in these same brain regions, comparable to those elicited by cocaine challenge [unpaired t-test: P = .66 (M1), P = .56 (dStr)]. However, the pO2 changes accompanying the hypercapnia challenge were substantially and significantly greater than those induced by cocaine administration (Fig. 3). In the motor cortex, ypO2 was 0.87F0.14 ( P b.002, unpaired t-test with ypO2 following cocaine), and in the striatum ypO2 was 1.36F0.18 ( P b.002, unpaired t-test with ypO2 following cocaine).
Fig. 3. Fractional changes in LDF and tissue pO2 following cocaine and hypercapnia challenge in (A) M1 and (B) dorsal striatum.
L. Ceolin et al. / Magnetic Resonance Imaging 25 (2007) 795 – 800
experiments, thus permitting a direct comparison of the temporal profiles of LDF, pO2 and rCBV data. Interestingly, LDF, pO2 and rCBV appear to be tightly coupled temporally in the early part of the response in all regions examined. However, in the motor cortex, the rCBV response was more sustained than that of LDF, suggesting a decoupling of these haemodynamic parameters at later time points in this brain region. We have recently demonstrated that regional differences in haemodynamic response to cocaine challenge cannot be attributed to different kinetics of the drug in plasma or tissues [3], nor to those of dopamine [3]. Various alternative hypotheses have been proposed, including regional differences in dopaminergic innervation or in the distribution of projections from other monoaminergic systems [2,22]. However, the mechanisms underlying these different temporal dynamics are still unknown. Cocaine challenge resulted in increased perfusion and tissue oxygen levels in all regions examined. Various authors have reported similar effects in the rat following brain activation by somatosensory stimulation [23–25]. However, elevated tissue oxygen levels are not an unambiguous signature of neuronal activation. In fact, increased perfusion may be driven by mechanisms that are independent of the underlying oxygen metabolism, like in the case of a CO2, and a mismatch between oxygen delivery and consumption can significantly alter tissue oxygen levels. Mild hypercapnia has been shown to increase perfusion without affecting cerebral oxidative metabolism and provides a calibration to assess the effects of drugs on oxygen metabolism. An important finding of this study is that cocaine induced much smaller changes in tissue oxygen levels compared to a CO2 challenge even though changes in LDF were comparable in the two experiments. This observation supports the hypothesis that cocaine affects tissue metabolism, thus increasing the local rate of oxygen consumption. A recent model of oxygen delivery to tissue proposed by Zheng et al. [14] provides a theoretical framework to illustrate this point (see Appendix A). Classic models of oxygen delivery, like the Buxton and Frank model [26,27], assume that tissue oxygen concentration is zero and does not vary during the haemodynamic response. Zheng et al. [14] have recently relaxed this assumption and have proposed a model that explicitly takes into account the buffering of oxygen by the tissue and the effects of tissue oxygen levels on the extraction of oxygen from the vasculature. When the modulatory effect of tissue oxygenation on oxygen transfer to the tissue is taken into account, the expression for the oxygen extraction fraction shows an explicit dependence on tissue oxygen levels (Eq. (4) of Appendix A), as well as on blood flow and perfusion. Under steady-state conditions, for a given value of blood flow and arterial blood oxygen saturation, lower levels of tissue oxygenation correspond to higher extraction fractions and, hence, to higher rates of
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oxygen metabolism. This is consistent with the independent finding that glucose utilization increases in several regions of the rat brain, including the striatum and cortex, upon cocaine challenge, as reported by Porrino et al. [21]. The present study shows that the haemodynamic response to cocaine challenge is accompanied by a concomitant change in oxygen metabolism. This evidence is at odds with the hypothesis that the phMRI response to cocaine may merely reflect vascular effects of the drugs. In conclusion, we have studied the changes in LDF and tissue oxygen tension in the rat brain elicited by 0.5 mg/kg acute intravenous cocaine challenge. The temporal profiles of these parameters were region dependent, consistent with previous observations. The LDF and pO2 responses to cocaine challenge were compared to those elicited by CO2 challenge. While changes in LDF were comparable in the two paradigms, the increase in tissular pO2 was significantly smaller in the cocaine protocol. These results support the idea that the haemodynamic response elicited by acute cocaine challenge is accompanied by an increase in oxygen consumption and that oxidative metabolism is a significant component of the metabolic response to cocaine.
Appendix A The rate of oxygen build-up in tissue can be described by the equation: V
dCt ¼ CMRO2 M dt
ð1Þ
where V is the blood volume, C t is the spatially averaged oxygen concentration in tissue, CMRO2 is the rate of oxygen delivery to tissue and M is the oxygen metabolic demand. A mismatch between the increases in oxygen delivery and oxygen consumption results in changes in oxygen oxygenation levels in tissue. At equilibrium: CMRO2 ¼ M
ð2Þ
i.e., all the oxygen extracted from the capillaries is metabolised. The rate of oxygen delivery to tissue can be expressed in terms of oxygen extraction fraction E: CMRO2 ¼ Cba EF C ba
ð3Þ
where is the arterial blood oxygen concentration and F is the blood flow entering the venous compartment. In the Buxton and Frank model, the oxygen extraction fraction is a function of blood flow only and is independent of tissue oxygenation. When the modulatory effect of tissue oxygenation is taken into account, the oxygen extraction fraction can be expressed as: " FF0 # E0 ð4Þ E ¼ ð1 g Þ 1 1 1 g0
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where g is the ratio of oxygen concentration in tissue (C t) to that of plasma in arterial blood (C pa), and the subscript 0 denotes the baseline value of each variable. Combination of Eqs. (1), (2) and (3) results in an expression for M that explicitly depends on tissue oxygenation: !" FF0 # Ct E0 a 1 1 F ð5Þ M ¼ Cb 1 a Cp 1 g0 Interestingly, for a given value F, lower levels of tissue oxygenation correspond to larger amounts of oxygen extracted from the vasculature and hence to higher rates of oxygen metabolism. References [1] Breiter HC, et al. Acute effects of cocaine on human brain activity and emotion. Neuron 1997;19:591 – 611. [2] Marota JJA, et al. Cocaine activation discriminates dopaminergic projections by temporal response: An fMRI study in rat. Neuroimage 2000;11:13 – 23. [3] Schwarz AJ, et al. Concurrent pharmacological MRI and in situ microdialysis of cocaine reveal a complex relationship between the central hemodynamic response and local dopamine concentration. Neuroimage 2004;23:296 – 304. [4] Stein EA, Fuller SA. Selective effects of cocaine on regional cerebral blood flow in the rat. J Pharmacol Exp Ther 1992;262:327 – 34. [5] Stein EA, Fuller SA. Cocaine’s time action profile on regional cerebral blood flow in the rat. Brain Res 1993;626:117 – 26. [6] Jenkins BG, Chen Y-CI, Mandeville JB. In: van Bruggen N, Roberts T, editors. Biomedical imaging in experimental neuroscience. New York7 CRC Press; 2003. p. 155 – 209. [7] Chen YC, et al. Detection of dopaminergic neurotransmitter activity using pharmacologic MRI: correlation with PET, microdialysis, and behavioral data. Magn Reson Med 1997;38:389 – 98. [8] Leslie RA, James MF. Pharmacological magnetic resonance imaging: a new application for functional MRI. Trends Pharmacol Sci 2000; 21:314 – 8. [9] Rudin M, et al. In vivo magnetic resonance methods in pharmaceutical research: current status and perspectives. NMR Biomed 1999; 12:69 – 97. [10] Koob GF, Le Moal M. Drug abuse: hedonic homeostatic dysregulation. Science 1997;278:52 – 8. [11] Kalisch R, Elbel GK, Gossl C, Czisch M, Auer DP. Blood pressure changes induced by arterial blood withdrawal influence bold signal in anesthesized rats at 7 tesla: implications for pharmacologic MRI. Neuroimage 2001;14:891 – 8.
[12] Luo F, Wu G, Li Z, Li S-J. Characterisation of effects of mean arterial blood pressure induced by cocaine and cocaine methiodide on BOLD signals in the rat brain. Magn Reson Med 2003;49:264 – 70. [13] Krimer LS, Muly III EC, Williams GV, Goldman-Rakic PS. Dopaminergic regulation of cerebral cortical microcirculation. Nat Neurosci 1998;1:286 – 9. [14] Zheng Y, et al. A model of the hemodynamic response and oxygen delivery to brain. Neuroimage 2002;16:617 – 37. [15] Nioka S, et al. Relationship between intracellular pH and energy metabolism in dog brain as measured by 31P-NMR. J Appl Physiol 1987;62:2094 – 102. [16] Davis TL, Kwong KK, Weisskoff RM, Rosen BR. Calibrated functional MRI: mapping the dynamics of oxidative metabolism. Proc Natl Acad Sci U S A 1998;95:1834 – 9. [17] Kim SG, Rostrup E, Larsson HB, Ogawa S, Paulson OB. Determination of relative CMRO2 from CBF and BOLD changes: significant increase of oxygen consumption rate during visual stimulation. Magn Reson Med 1999;41:1152 – 61. [18] Hoge RD, et al. Investigation of BOLD signal dependence on cerebral blood flow and oxygen consumption: the deoxyhemoglobin dilution model. Magn Reson Med 1999;42:849 – 63. [19] Griffiths JR, Robinson SP. The OxyLite: a fibre-optic oxygen sensor. Br J Radiol 1999;72:627 – 30. [20] Braun RD, Lanzen JL, Snyder SA, Dewhirst MW. Comparison of tumor and normal tissue oxygen tension measurements using OxyLite or microelectrodes in rodents. Am J Physiol Heart Circ Physiol 2001;280:H2533. [21] Porrino LJ, Domer FR, Crane AM, Sokoloff L. Selective alterations in cerebral metabolism within the mesocorticolimbic dopaminergic system produced by acute cocaine administration in rats. Neuropsychopharmacology 1988;1:109 – 18. [22] Barinaga M. What makes brain neurons run? Science 1997;276:196 – 8. [23] Ances BM, Buerk DG, Greenberg JH, Detre JA. Temporal dynamics of the partial pressure of brain tissue oxygen during functional forepaw stimulation in rats. Neurosci Lett 2001;306:106 – 10. [24] Ances BM, Wilson DF, Greenberg JH, Detre JA. Dynamic changes in cerebral blood flow, O2 tension, and calculated cerebral metabolic rate of O2 during functional activation using oxygen phosphorescence quenching. J Cereb Blood Flow Metab 2001;21:511 – 6. [25] Buerk DG, Ances BM, Greenberg JH, Detre JA. Temporal dynamics of brain tissue nitric oxide during functional forepaw stimulation in rats. Neuroimage 2003;18:1 – 9. [26] Buxton RB, Frank LR. A model for the coupling between cerebral blood flow and oxygen metabolism during neural stimulation. J Cereb Blood Flow Metab 1997;17:64 – 72. [27] Buxton RB, Wong EC, Frank LR. Dynamics of blood flow and oxygenation changes during brain activation: the balloon model. Magn Reson Med 1998;39:855 – 64. [28] Paxinos G, Watson C. The rat brain in stereotaxic coordinates. San Diego7 Academic Press; 1998.
Effects of cocaine on blood flow and oxygen metabolism in the rat brain: implications for phMRI Laura Ceolin, Adam J. Schwarz, Alessandro Gozzi, Torsten Reese, Angelo Bifone4 Department of Neuroimaging, Psychiatry CEDD, GlaxoSmithKline Medicines Research Centre, 37135 Verona, Italy Accepted 11 January 2007
Abstract The effects of cocaine on cerebral blood flow and tissue oxygen levels in the rat brain were investigated with concurrent laser Doppler flowmetry and fluorescence quenching spectroscopy. Responses elicited by mild hypercapnia were used as calibration to assess the effects of cocaine on oxidative metabolism. Intravenous cocaine challenge of 0.5 mg/kg induced significant increases in tissular oxygenation and perfusion in all regions investigated (primary motor cortex, medial prefrontal cortex and dorsal striatum). Mild hypercapnia, a challenge that affects haemodynamics but not metabolism, elicited comparable changes in blood flow but substantially larger changes in tissue oxygen levels. These differences in tissue oxygen build-up suggest that increased oxidative metabolism is a significant component of the cerebral metabolic response to acute cocaine challenge. The implications for the interpretation of pharmacological MRI data are discussed. D 2007 Elsevier Inc. All rights reserved. Keywords: Cocaine; Functional MRI; fMRI; phMRI; Brain metabolism; Oxygen consumption; LDF; CBF
1. Introduction Functional magnetic resonance imaging (fMRI) methods have been applied to map the central haemodynamic response to cocaine challenge as a surrogate for changes in brain activity in humans [1] and laboratory animals [2–5]. This fMRI approach, sometimes referred to as bpharmacological MRIQ (phMRI) [6–9] is promising for the investigation of the central activity of cocaine and other psychoactive drugs at a system level. However, the complex effects of cocaine on neuronal activity, brain metabolism and vascular response are not fully understood, and a link between haemodynamic changes and the underlying neuronal activity remains to be demonstrated. One of the primary actions of cocaine in the brain is blockade of dopamine transporters, resulting in an increase in synaptic dopamine levels. These modulations in the mesolimbic dopamine pathway have been associated with the psychostimulant effects and reinforcing properties of cocaine [10]. Conceivably, changes in neuronal activity induced by cocaine might induce changes in brain metabolism, which in turn might trigger a central haemodynamic 4 Corresponding author. Tel.: +39 045 921 9707. E-mail address: [email protected] (A. Bifone). 0730-725X/$ – see front matter D 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.mri.2006.10.022
response. Indeed, acute cocaine challenge in the rat induces a widespread central haemodynamic response whose amplitude and temporal profile vary with anatomical location [2,3]. However, other mechanisms have been invoked, including changes in blood pressure [11,12], direct vasoactivity of dopamine [7,13] and innervation of blood vessels by dopaminergic neurons [13]. Recently, we have investigated the correlation between dopamine levels and relative cerebral blood volume (rCBV) changes in the rat brain by concurrent microdialysis and fMRI [3]. Interestingly, an fMRI response was observed also in brain structures, like the motor cortex, where cocaine did not elicit detectable changes in dopamine levels, thus indicating that in these areas the haemodynamic response is not directly driven by local dopamine modulations. Here we have investigated the effects of cocaine on brain tissue oxygen levels and their correlation with haemodynamic changes elicited by the drug. Specifically, we have applied fluorescence quenching spectroscopy and laser Doppler flowmetry (LDF) to simultaneously measure brain tissue oxygen tension (pO2) and cerebral blood flow following acute cocaine challenge in the anaesthetised rat. Changes in tissue pO2 reflect transient imbalance between oxygen consumption and supply, and are sensitive to local changes in oxygen metabolic rate [14]. Three different brain
796
L. Ceolin et al. / Magnetic Resonance Imaging 25 (2007) 795 – 800
regions, the primary motor cortex (M1), medial prefrontal cortex (mPFC) and dorsal striatum (dStr) were investigated. These regions were chosen because each shows a different temporal rCBV response profile [2] and a different relationship with local changes in dopamine levels as detected by microdialysis [3]. For comparison, we have also measured the response to CO2 challenge in the primary motor cortex and in dStr. Mild hypercapnia has been shown to induce a haemodynamic response without significantly affecting brain metabolism [15] and has been proposed as a calibration to measure changes in oxygen metabolic rates [16–18]. The aim of the present study was twofold: (a)
to measure the temporal dynamics of tissue oxygen levels and blood flow following cocaine challenge; (b) to investigate the correlation between haemodynamic response elicited by cocaine and the underlying changes in oxygen metabolism. 2. Materials and methods 2.1. The Oxylite system A combined Oxylite/LDF system (Oxford Optronix, Oxford, UK) was used to simultaneously measure tissue oxygen tension and blood flow in the rat brain. Oxylite technology uses a ruthenium luminophor located at the tip of an optical fiber, which conveys light pulses generated by a diode to the photoactive material. The lifetime of the resultant fluorescence is inversely proportional to pO2 at the probe tip. The diameter of the optical fiber is 220 Am. The Oxylite probe is bundled with another optical fiber (200 Am diameter) for the laser Doppler flow measurement. The tips of the probes are closely positioned to provide simultaneous measurements of the two parameters over approximately the same volume. All probes were precalibrated by the manufacturer, and the calibration parameters were scanned into the system prior to each experiment. Oxylite presents several advantages over polarographic measurements of pO2 for the scope of this experiment. Firstly, Oxylite does not consume oxygen during the measurement and allows continuous monitoring of oxygen levels for extended periods of time [19]. Moreover, the Oxylite probe measures an average pO2 over a sensing volume that is substantially larger than that probed by microelectrodes [20]. Thus, Oxylite is less sensitive to the heterogeneity of oxygen concentration on the length scale of capillaries. 2.2. Animal preparation All experiments were carried out in strict accordance with Italian animal welfare legislation and GSK internal ethical review. Male Sprague-Dawley rats (Charles River, Italy), weighing 250–350 g, were anesthetized with 3% halothane in a 30%/70% O2/N2 mixture using a facemask. The animals were tracheotomised and artificially ventilated with a
mechanical respirator (Inspira, Oxford, UK). Upon tracheotomy and throughout surgery, the anaesthetic level was maintained at 1.5–2.0%. Before each incision, the surgical area was treated subcutaneously with 0.3 ml marcaine, a topical anesthetic. The left femoral artery and vein were cannulated with a PE50 polyethylene catheter for the infusion of compounds and for monitoring of arterial blood pressure. Subsequently, the animals were placed in a stereotaxic frame for the insertion of the combined dual fluorescence-quenching/laser Doppler flow probe. The animals were divided into three groups, with the combined Oxylite/LDF probe located in either the primary motor cortex, dStr or medial prefrontal cortex. A hole was drilled through the parietal bone, and the probe was inserted perpendicular to the brain surface according to the coordinates from dura mater [28] as follows: motor cortex (M1) AP +2.2 mm, ML +2.8 mm, DV 2.5 mm; dStr AP +1.0 mm, ML +2.5 mm, DV 4.1 mm; mPFC AP +2.7 mm, ML +0.5 mm, DV 3.5 mm. After surgery, the halothane concentration was decreased to a maintenance level of 1%. d-Tubocurarine (0.25 mg/kg h iv, Sigma-Aldrich, Milan, Italy), dissolved in saline heparin (25 UI/ml), was infused through the cannulated artery to maintain muscle relaxation. Mean arterial blood pressure and heart rate were monitored continually throughout the experiment with a blood pressure transducer (Biopac Systems Corp., Goweta, USA) connected to the arterial catheter. The body temperature was monitored with a rectal probe and maintained at 37.5F18C using a heating pad. The volume of ventilation was adjusted to maintain blood pCO2 within physiological range and blood pO2 N 100. A period of stabilisation of 2 h was allowed before starting the pO2/LDF measurement. Blood samples of ca. 200 Al were collected from the arterial catheter before and immediately after the end of pO2/LDF data acquisition, and blood gas levels measured using a blood gas analyzer (AVL, Roswell, GA, USA). The protocol of the present study, including anaesthetic regimen, animal preparation, monitoring and stereotaxic coordinates, was very similar to that we used in a previously published phMRI/microdialysis study [3] in order to facilitate comparison of cocaine-induced pO2 and LDF changes with the rCBV response. Three groups of animals were infused intravenously over 1 min with either a cocaine challenge (Sigma, 0.5 mg/kg in saline, 1.0 ml/rat) or vehicle (saline, 1.0 ml/kg). Group sizes were as follows: M1, n = 7 (5 cocaine, 2 saline); dStr, n = 10 (4 cocaine, 6 saline); mPFC, n = 11 (6 cocaine, 5 saline). A dose of 0.5 mg/kg of cocaine was chosen for consistency with previous phMRI [2,3], microdialysis [3] and deoxyglucose autoradiography studies [21]. Two separate groups of animals (M1 and dStr, n = 4 each) were challenged with CO2 through the mechanical ventilation system. In order to determine the concentration of CO2 that best mimicked the LDF response to cocaine, different amounts of CO2 (in the range 0.05 to 0.1 l per minute, corresponding to 2.5–5% CO2) were added to the O2/N2
L. Ceolin et al. / Magnetic Resonance Imaging 25 (2007) 795 – 800
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Fig. 1. Temporal profiles of LDF, pO2 and MBP changes following cocaine and vehicle (saline) challenge measured in (A) the motor cortex (M1), (B) the dorsal striatum and (C) the mPFC.
mixture for periods of 10 min, followed by 10 min of recovery. Basal blood pO2 and pCO2 levels were in the range 180–210 and 34–37 mm Hg, respectively, for all groups. No significant changes in blood gas levels were observed after cocaine challenge. In the subjects challenged with CO2, no significant changes in pO2 were observed, while pCO2 levels rose slightly, but did not exceed 41 mm Hg, well within the range of mild hypercapnia conditions. Tissue pO2 and LDF values were measured for 10 min before and 30 min after vehicle, cocaine or CO2 challenge. At the end of the experiment, the animals were euthanized with an overdose of anaesthetic followed by cervical dislocation. 2.3. Data analysis Time courses for MBP, pO2 and LDF were exported from the Biopac software at a time resolution of ~10 s per data point. Data were further rebinned to 1-min intervals for the time-course figures for clarity of display. In order to compare changes in tissular pO2 for similar changes in local LDF between cocaine- and hypercapnia-challenged animals, each time course was normalised to the mean of a 10-min baseline period prior to the challenge and thus expressed as a fractional change, denoted yLDF and ypO2, respectively. For each animal, the average yLDF and ypO2 over a 3-min time window at the peak response was used as a summary
statistic of the relative change in each parameter. This captured stable LDF and pO2 values corresponding to a steady-state regime. Statistical analysis was performed on the fractional changes. For the hypercapnia-challenged animals, the CO2 challenge (0.05 or 0.1 l per minute) that yielded a yLDF closest to the mean yLDF in the corresponding cocaine group was used, so that the LDF changes matched as closely as possible those arising following cocaine challenge. The values of yLDF and ypO2 were compared between cocaineand hypercapnia-challenged groups in the striatum and motor cortex by two-sample T-test (Statistica v. 6.1, StatSoft Inc.). Threshold for statistical significance was considered as P =.05. Results are quoted and shown as meanFS.E.M. unless otherwise indicated.
3. Results 3.1. Temporal profiles The temporal profiles of LDF, pO2 and mean blood pressure (MBP) changes following acute cocaine challenge are shown in Fig. 1. In the motor cortex, both LDF and pO2 showed a rapid increase to a peak about 4 min postinjection and returned to baseline values within ~15 min. In the striatum, LDF and pO2 responses were more sustained, with the tail of the response more variable between subjects, as
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Fig. 2. Temporal profiles of LDF, pO2 and MBP changes following hypercapnia challenge measured in (A) the motor cortex (M1) and (B) the dorsal striatum.
reflected by the larger error bars at late time points. In both brain regions, the time courses of LDF and pO2 changes were positively correlated (mean intra-animal cross-correlation: q =0.66). In the mPFC, LDF changes were more sustained than for pO2, which rose to a broad maximum after 5–6 min, before returning more slowly toward baseline values after ~30 min. Vehicle injection did not produce significant LDF or pO2 changes, and neither cocaine nor saline significantly affected blood pressure. The temporal profiles of LDF, pO2 and MBP following CO2 challenge are shown in Fig. 2. In the motor cortex, LDF rose rapidly, reached its peak value within 2–3 min and remained sustained over the duration of the experiment. Tissue oxygen tension rose more slowly and reached a plateau after 6–7 min from the beginning of the CO2 challenge. In the dStr, LDF peaked after 4 min and slowly decreased towards baseline, while pO2 reached a plateau. Transient negative changes in arterial blood pressure were observed following CO2 challenge, but they did not reach statistical significance.
4. Discussion and conclusion The temporal profiles of LDF and pO2 following cocaine challenge were positively correlated in all the regions examined in this study. Interestingly, the LDF and pO2 time courses varied with anatomical location, with a faster response in the motor cortex and more sustained responses in the mPFC and in the dStr. This observation is consistent with previous rCBV measurements [2,3]. The rCBV responses reported in Ref. [3], in particular, were acquired under very similar experimental conditions and animal preparation procedures to those used in the present
3.2. Amplitude changes in LDF and pO2 Cocaine challenge elicited peak fractional LDF changes of 0.24F0.08 and 0.29F0.16 in M1 and dStr, respectively. These were accompanied by increases in tissue pO2 of similar magnitude, 0.28F0.03 and 0.29F0.13, respectively (Fig. 3). Hypercapnia challenge yielded LDF changes of 0.30F0.14 and 0.20F0.05 in these same brain regions, comparable to those elicited by cocaine challenge [unpaired t-test: P = .66 (M1), P = .56 (dStr)]. However, the pO2 changes accompanying the hypercapnia challenge were substantially and significantly greater than those induced by cocaine administration (Fig. 3). In the motor cortex, ypO2 was 0.87F0.14 ( P b.002, unpaired t-test with ypO2 following cocaine), and in the striatum ypO2 was 1.36F0.18 ( P b.002, unpaired t-test with ypO2 following cocaine).
Fig. 3. Fractional changes in LDF and tissue pO2 following cocaine and hypercapnia challenge in (A) M1 and (B) dorsal striatum.
L. Ceolin et al. / Magnetic Resonance Imaging 25 (2007) 795 – 800
experiments, thus permitting a direct comparison of the temporal profiles of LDF, pO2 and rCBV data. Interestingly, LDF, pO2 and rCBV appear to be tightly coupled temporally in the early part of the response in all regions examined. However, in the motor cortex, the rCBV response was more sustained than that of LDF, suggesting a decoupling of these haemodynamic parameters at later time points in this brain region. We have recently demonstrated that regional differences in haemodynamic response to cocaine challenge cannot be attributed to different kinetics of the drug in plasma or tissues [3], nor to those of dopamine [3]. Various alternative hypotheses have been proposed, including regional differences in dopaminergic innervation or in the distribution of projections from other monoaminergic systems [2,22]. However, the mechanisms underlying these different temporal dynamics are still unknown. Cocaine challenge resulted in increased perfusion and tissue oxygen levels in all regions examined. Various authors have reported similar effects in the rat following brain activation by somatosensory stimulation [23–25]. However, elevated tissue oxygen levels are not an unambiguous signature of neuronal activation. In fact, increased perfusion may be driven by mechanisms that are independent of the underlying oxygen metabolism, like in the case of a CO2, and a mismatch between oxygen delivery and consumption can significantly alter tissue oxygen levels. Mild hypercapnia has been shown to increase perfusion without affecting cerebral oxidative metabolism and provides a calibration to assess the effects of drugs on oxygen metabolism. An important finding of this study is that cocaine induced much smaller changes in tissue oxygen levels compared to a CO2 challenge even though changes in LDF were comparable in the two experiments. This observation supports the hypothesis that cocaine affects tissue metabolism, thus increasing the local rate of oxygen consumption. A recent model of oxygen delivery to tissue proposed by Zheng et al. [14] provides a theoretical framework to illustrate this point (see Appendix A). Classic models of oxygen delivery, like the Buxton and Frank model [26,27], assume that tissue oxygen concentration is zero and does not vary during the haemodynamic response. Zheng et al. [14] have recently relaxed this assumption and have proposed a model that explicitly takes into account the buffering of oxygen by the tissue and the effects of tissue oxygen levels on the extraction of oxygen from the vasculature. When the modulatory effect of tissue oxygenation on oxygen transfer to the tissue is taken into account, the expression for the oxygen extraction fraction shows an explicit dependence on tissue oxygen levels (Eq. (4) of Appendix A), as well as on blood flow and perfusion. Under steady-state conditions, for a given value of blood flow and arterial blood oxygen saturation, lower levels of tissue oxygenation correspond to higher extraction fractions and, hence, to higher rates of
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oxygen metabolism. This is consistent with the independent finding that glucose utilization increases in several regions of the rat brain, including the striatum and cortex, upon cocaine challenge, as reported by Porrino et al. [21]. The present study shows that the haemodynamic response to cocaine challenge is accompanied by a concomitant change in oxygen metabolism. This evidence is at odds with the hypothesis that the phMRI response to cocaine may merely reflect vascular effects of the drugs. In conclusion, we have studied the changes in LDF and tissue oxygen tension in the rat brain elicited by 0.5 mg/kg acute intravenous cocaine challenge. The temporal profiles of these parameters were region dependent, consistent with previous observations. The LDF and pO2 responses to cocaine challenge were compared to those elicited by CO2 challenge. While changes in LDF were comparable in the two paradigms, the increase in tissular pO2 was significantly smaller in the cocaine protocol. These results support the idea that the haemodynamic response elicited by acute cocaine challenge is accompanied by an increase in oxygen consumption and that oxidative metabolism is a significant component of the metabolic response to cocaine.
Appendix A The rate of oxygen build-up in tissue can be described by the equation: V
dCt ¼ CMRO2 M dt
ð1Þ
where V is the blood volume, C t is the spatially averaged oxygen concentration in tissue, CMRO2 is the rate of oxygen delivery to tissue and M is the oxygen metabolic demand. A mismatch between the increases in oxygen delivery and oxygen consumption results in changes in oxygen oxygenation levels in tissue. At equilibrium: CMRO2 ¼ M
ð2Þ
i.e., all the oxygen extracted from the capillaries is metabolised. The rate of oxygen delivery to tissue can be expressed in terms of oxygen extraction fraction E: CMRO2 ¼ Cba EF C ba
ð3Þ
where is the arterial blood oxygen concentration and F is the blood flow entering the venous compartment. In the Buxton and Frank model, the oxygen extraction fraction is a function of blood flow only and is independent of tissue oxygenation. When the modulatory effect of tissue oxygenation is taken into account, the oxygen extraction fraction can be expressed as: " FF0 # E0 ð4Þ E ¼ ð1 g Þ 1 1 1 g0
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where g is the ratio of oxygen concentration in tissue (C t) to that of plasma in arterial blood (C pa), and the subscript 0 denotes the baseline value of each variable. Combination of Eqs. (1), (2) and (3) results in an expression for M that explicitly depends on tissue oxygenation: !" FF0 # Ct E0 a 1 1 F ð5Þ M ¼ Cb 1 a Cp 1 g0 Interestingly, for a given value F, lower levels of tissue oxygenation correspond to larger amounts of oxygen extracted from the vasculature and hence to higher rates of oxygen metabolism. References [1] Breiter HC, et al. Acute effects of cocaine on human brain activity and emotion. Neuron 1997;19:591 – 611. [2] Marota JJA, et al. Cocaine activation discriminates dopaminergic projections by temporal response: An fMRI study in rat. Neuroimage 2000;11:13 – 23. [3] Schwarz AJ, et al. Concurrent pharmacological MRI and in situ microdialysis of cocaine reveal a complex relationship between the central hemodynamic response and local dopamine concentration. Neuroimage 2004;23:296 – 304. [4] Stein EA, Fuller SA. Selective effects of cocaine on regional cerebral blood flow in the rat. J Pharmacol Exp Ther 1992;262:327 – 34. [5] Stein EA, Fuller SA. Cocaine’s time action profile on regional cerebral blood flow in the rat. Brain Res 1993;626:117 – 26. [6] Jenkins BG, Chen Y-CI, Mandeville JB. In: van Bruggen N, Roberts T, editors. Biomedical imaging in experimental neuroscience. New York7 CRC Press; 2003. p. 155 – 209. [7] Chen YC, et al. Detection of dopaminergic neurotransmitter activity using pharmacologic MRI: correlation with PET, microdialysis, and behavioral data. Magn Reson Med 1997;38:389 – 98. [8] Leslie RA, James MF. Pharmacological magnetic resonance imaging: a new application for functional MRI. Trends Pharmacol Sci 2000; 21:314 – 8. [9] Rudin M, et al. In vivo magnetic resonance methods in pharmaceutical research: current status and perspectives. NMR Biomed 1999; 12:69 – 97. [10] Koob GF, Le Moal M. Drug abuse: hedonic homeostatic dysregulation. Science 1997;278:52 – 8. [11] Kalisch R, Elbel GK, Gossl C, Czisch M, Auer DP. Blood pressure changes induced by arterial blood withdrawal influence bold signal in anesthesized rats at 7 tesla: implications for pharmacologic MRI. Neuroimage 2001;14:891 – 8.
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