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A multimodality investigation of cerebral hemodynamics and autoregulation in pharmacological MRI

A multimodality investigation of cerebral hemodynamics and autoregulation in pharmacological MRI
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  A multimodality investigation of cerebral hemodynamics andautoregulation in pharmacological MRI Alessandro Gozzi a, 4 , Laura Ceolin a  , Adam Schwarz a  , Torsten Reese a  , Simone Bertani  b ,Valerio Crestan  b , Angelo Bifone a  a   Department of Neuroimaging, Centre of Excellence for Drug Discovery, Psychiatry, GlaxoSmithKline Medicines Research Centre, 37135 Verona, Italy  b  Laboratory Animal Science, Centre of Excellence for Drug Discovery, Psychiatry, GlaxoSmithKline Medicines Research Centre, 37135 Verona, Italy Accepted 11 January 2007 Abstract Pharmacological MRI (phMRI) methods have been widely applied to assess the central hemodynamic response to pharmacologicalintervention as a surrogate for changes in the underlying neuronal activity. However, many psychoactive drugs can also affect cardiovascular  parameters, including arterial blood pressure (BP). Abrupt changes in BP or the anesthetic agents used in preclinical phMRI may impair cerebral blood flow (CBF) autoregulation mechanisms, potentially introducing confounds in the phMRI response. Moreover, relative cerebral blood volume (rCBV), often measured in small-animal phMRI studies, may be sensitive to BP changes even in the presence of intact autoregulation. We applied laser Doppler flowmetry and MRI to measure changes in CBF and microvascular CBV induced by increasingdoses of intravenous norepinephrine (NE) challenge in the halothane-anesthetized rat. NE is a potent vasopressor that does not cross the blood–brain barrier and mimics the rapid BP changes typically observed with acute drug challenges. We found that CBF autoregulation wasmaintained over a BP range of 60–120 mmHg. Under these conditions, no significant central rCBV responses were observed, suggesting that microvascular rCBV changes in response to abrupt changes in perfusion pressure are negligible within the autoregulatory range. Larger BPresponses were accompanied by significant changes in both CBV and CBF that might confound the interpretation of phMRI results. D  2007 Elsevier Inc. All rights reserved.  Keywords:  phMRI; CBF; CBV; Autoregulation; Rat; Blood pressure 1. Introduction Pharmacological MRI (phMRI) methods can be appliedto assess the effects of acute drug challenge on cerebralhemodynamics as a surrogate for changes in the underlyingneuronal activity. This approach has been widely applied tostudy central effects of drugs on the central nervous system(CNS) in humans and animal models [1,2]. However, many of these drugs can also induce significant peripheral effects,including severe alterations of cardiovascular parameters.Under physiological conditions, mechanisms of autoregula-tion keep cerebral blood flow (CBF) relatively constant inthe presence of changes in mean arterial blood pressure(MABP). However, general anesthetics, widely used in preclinical phMRI studies to avoid head motion and to better control animal physiology, may affect the centralvasoadaptive response to peripheral MABP changes, thusmaking it difficult to predict the influence of systemicvasopressive effects on cerebral hemodynamics. Moreover,large and rapid changes in MABP may cause a breakdownin the autoregulatory mechanisms that control brainmicrocirculation, thus introducing potential confounds inthe interpretation of phMRI data.While blood-oxygen-level-dependent (BOLD) signalsare most often measured in humans, relative cerebral bloodvolume (rCBV) has been widely used in phMRI studies insmall laboratory animals due to the increased sensitivityafforded by rCBV measurements with intravascular contrast agents over BOLD [3]. However, dilation and constriction of cerebral blood vessels are thought to modulate vascular resistance in order to maintain CBF relatively constant in the presence of changes in perfusion pressure [4]. As aconsequence, CBV might be sensitive to MABP changeseven in the presence of intact autoregulation.Several attempts to correlate the magnitude of sys-temic MABP changes with the central hemodynamic res- 0730-725X/$ – see front matter   D  2007 Elsevier Inc. All rights reserved.doi:10.1016/j.mri.2007.03.003 4  Corresponding author. Tel.: +39 0458219233; fax: +39 0458218073.  E-mail address: (A. Gozzi).Magnetic Resonance Imaging 25 (2007) 826–833   ponses in the rodent brain have been published. Zaharchuk et al. [5] did not observe significant CBF, CBV or BOLD changes as MABP was gradually decreased(  1 mmHg/min) by continuous arterial blood withdrawalover the range of maximally effective autoregulation in thehalothane-anesthetized rat. However, in a more recent study, Kalisch et al. [6] argued that slow and gradual decreases in MABP may not be representative of theabrupt changes typically observed in pharmacological MR experiments. Indeed, the same authors reported a signifi-cant correlation between BOLD signal time courses andMABP changes following rapid arterial blood withdrawal– reinfusion under three different anesthetic regimes in therat (isoflurane, halothane and propofol). However, the useof the blood withdrawal–reinfusion method presents potential drawbacks such as the need to account for hemodilution, the risk of hemorrhagic shock-like compli-cations and the lack of a stable and normotensive prestimulus MABP baseline.Other investigators have measured the BOLD signalchanges produced by pharmacologically evoked MABPalterations. Tuor et al. [7] and, more recently, Wang et  al. [8] reported significant correlations between BOLD signal andthe MABP changes induced by norepinephrine (NE), a non- brain-penetrant vasopressor, in rats anesthetized with  a -chloralose. Luo et al. [9] observed significant fMRIresponses under urethane anesthesia following acute chal-lenge with cocaine but not with cocaine methiodide, a non- brain-penetrant cocaine analogue, at doses that increasedMABP up to 180 mmHg, thus suggesting that potentiallyconfounding peripheral effects were negligible in that specific protocol. However, the BOLD response may result from changes in several metabolic and hemodynamic parameters whose contributions cannot be easily disen-tangled, and these conclusions cannot be extended to phMRI methods based on rCBV measurements.Here, we have applied laser Doppler flowmetry (LDF)and MRI to measure changes in CBF and microvascular CBV induced by increasing doses of an intravenous NEchallenge in the halothane-anesthetized rat. The rCBV protocol employed has been used by us as well as by other groups to map the central hemodynamic response to anumber  of neuroactive compounds, including am phetamine [10–13], cocaine [14,15], apomorphine [16,17] and nico- tine [18,19]. Following Tuor’s approach, we explored increasing doses of NE in order to correlate the magnitudeof the cardiovascular response with the correspondingchanges in CBV, to assess the potentially confoundingeffects of MABP changes on CBV-based phMRI data. Theuse of a pharmacological vasopressor has the advantage of circumventing the limitations of the blood withdrawal andreinfusion method while reproducing the abrupt MABPchanges that are typically observed upon drug injection. Bymeasuring LDF changes, we were able to assess theintegrity of CBF autoregulatory mechanisms in our model.The independent measurement of CBF and CBV enabledus to investigate the interplay of these two parameters inthe range of effective vasoadaptive response and under autoregulation breakdown. 2. Methods 2.1. Animal preparation All experiments were carried out in accordance withItalian regulations governing animal welfare and protection.Protocols were also reviewed and consented to by a localanimal care committee, in accordance with the guidelinesof the  Principles of Laboratory Animal Care  (NIH publication 86-23, revised 1985). These studies were performed on male Sprague–Dawley rats (250–350 g;Charles River, Como, Italy). Animals had free access tostandard rat chow and tap water and were housed in groupsof five in solid bottom cages with sawdust litter. Roomtemperature (20–22 8 C), relative humidity (45–65%) anddark–light cycles (12 h each, lights on at 0600 h) wereautomatically controlled. After arrival, rats were allowed toacclimatize for at least 5 days. 2.2. rCBV measurements Animal preparation/monitoring and MRI acquisition ineach phMRI study were similar to those in previous studies[18]. Briefly, rats were anesthetized with 3% halothane in a30%:70% O 2 :N 2  gas mixture, tracheotomized and artifi-cially ventilated with a mechanical respirator. The ventila-tion volume was adjusted to maintain physiological levelsof   p a  O 2  and  p a  CO 2  according to arterial blood gases’measurements performed during the study. The left femoralartery and vein were cannulated, and the animal was paralyzed with a 0.25-mg/kg iv bolus of   d -tubocurarinefollowed by a continuous infusion of 0.25 mg/kg/h throughthe artery. All wounds were infiltrated with 1% lidocaine before incision. After surgery, the rat was secured into acustomized stereotaxic holder (Bruker, Ettlingen, Ger-many), and the halothane level was set to 1% (1 MAC[20]). An MR-compatible thermocouple probe was used tomeasure rectal temperature. The body temperature of allsubjects remained within physiological range (37 F 1.5 8 C).MABP was monitored continually throughout the MRIexperiment. At the end of the experiment, the animals wereeuthanized with an overdose of anesthetic followed bycervical dislocation.MRI data were acquired using a Bruker Avance 4.7-Tsystem, a 72-mm birdcage resonator for RF transmit and aBruker curved  b Rat Brain  Q   quadrature receive coil. TheMR acquisition for each subject comprised RARE  T  2 -weighted anatomical images (TR  eff  , 5000 ms; TE eff  , 76 ms;RARE factor, 32; FOV, 40 mm; 256  256 matrix; 16contiguous 1-mm slices) followed by a time seriesacquisition with the same spatial coverage and similar  parameters (TR  eff  , 2700 ms; TE eff  , 110 ms), but with alower in-plane spatial resolution (128  128), giving afunctional pixel volume of ~0.1 mm 3 . Following five  A. Gozzi et al. / Magnetic Resonance Imaging 25 (2007) 826–833  827  reference images, 2.67 ml/kg of the blood-pool contrast agent Endorem (Guerbet, France) was injected so that subsequent signal changes would reflect alterations inrCBV [21,22]. Four successive scans were averaged for a resulting time resolution of 80 s. Signal intensity changesin the time series were converted into fractional rCBV ona pixel-wise basis, using a constrained exponential modelof the gr adual elimination of contrast agent from the blood pool [23].The rCBV time series data for each experiment wereanalyzed on both an image and volume of interest (VOI) basis within the framework of the general linear model.Individual subjects in each study were spatially normal-ized by a 9- df    affine transformation, mapping their   T  2 -weighted anatomical images to a stereotaxic rat brain MRItemplate set  [16] and applying the resulting transformation matrix to the accompanying rCBV time series (FSL/ FLIRT v.5.2). RCBV time series were calculated coveringa 6-min 40-s (10 time points) preinjection baseline and12 min postinjection (18 time points), normalized to acommon injection time point. This time window capturedthe relatively rapid rCBV signal changes observedfollowing injection of NE (see Section 3). Image-basedtime series analysis was carried out using FEAT (fMRIExpert Analysis Tool) Version 5.43, part of FSL (FMRIB’sSoftware Library,, with a 0.6-mm spatial smoothing, and using a model function identified bywavelet cluster analysis across all animals in the cohort,capturing the temporal profile of the postinjection signalchange [24,25]. The design matrix also included the temporal derivative of this regressor and a linear ramp(both orthogonalized to the regressor of interest). Thecoefficients of the model function thus provided a map of rCBV response amplitude for each injection in each subject.Higher-level group comparisons were carried out usingordinary least squares simple mixed effects;  Z   (Gaussianized T  /   F  ) statistic images were thresholded using clustersdetermined by  Z  N 2.3 and a (corrected) cluster significancethreshold of   P  =.05 [26].VOI time courses were extracted from unsmoothedrCBV time series data using a 3D digital reconstruction of a rat brain atlas [27] coregistered with the MRI template[16], using custom in-house software written in IDL(Research Systems Inc., Boulder, CO). For each VOI timecourse, the average rCBV over a 4-min time windowcovering the peak response (from 1 min 20 s to 5 min 20 s postinjection; 7 time points) was used as a summary statisticof the relative change. The statistical significance of NE’seffects was assessed versus the postinjection response invehicle-treated animals, as this reflects standard practicein phMRI. Group rCBV response from VOIs was compared between vehicle and NE-challenged groups by a one-way ANOVA followed by a Dunnett’s test for multiplecomparisons. Threshold for statistical significance wasconsidered as  P  =.05. Results are quoted and displayed asmean F S.E.M. unless otherwise indicated. 2.3. LDF measurements An Oxylite/LDF system (Oxford Optronix, Oxford, UK)was used to measure blood flow in the rat brain. Oxylitetechnology uses an optical fiber (200  A m diameter) for theLDF measurement. All probes are precalibrated by themanufacturer, and the calibration parameters are scannedinto the system prior to each experiment. Animals were prepared using the same animal preparation protocoldescribed above for phMRI experiments. At the end of surgery, the animals were placed in a stereotaxic frame for the insertion of the LDF probe. A hole was drilled throughthe parietal bone, and the probe was inserted in the right S1FL cortex perpendicular to the brain surface using thefollowing coordinates from dura mater  [28]: AP, +2.2 mm; ML, +2.8 mm; DV,  2.5 mm. S1FL is a region that ensuresstable and reliable LDF measurements and in which highdoses of NE induced significant rCBV changes (see Section3). LDF and MABP were concurrently monitored through-out the experiment using a multichannel MP150 Biopacdata-acquisition system (Biopac Systems Inc., Goweta,USA). The body temperature was monitored with a rectal probe and maintained at 37.5 F 1 8 C using a heating pad. Theventilation volume was adjusted to maintain physiologicallevels of   p a  O 2  and  p a  CO 2  according to arterial blood gases’measurements performed during the study. LDF values wererecorded for 5 min before and 15 min after NE injection. At the end of the experiment, the animals were euthanized withan overdose of anesthetic followed by cervical dislocation. 2.4. Experiments and drugs RCBVand LDF were measured in two separate groups of animals. In the rCBV experiment, after 20 min of stabilization, animals were intravenously challenged withincreasing doses of NE [ l -(  )-norepinephrine-(+)-bitartrate,CALBIOCHEM; 0.125  A g/kg ( n =5), 0.5  A g/kg ( n =5),2  A g/kg ( n =5) or 8  A g/kg ( n =5)] or its vehicle (saline; n =5). In the LDF study, animals were challenged with NEafter 2 h of stabilization. In this case, group sizes were asfollows: vehicle ( n =4), 0.125  A g/kg ( n =4), 0.5  A g/kg( n =4), 2  A g/kg ( n =4) or 8  A g/kg ( n =4). NE doses refer tothe salt form of the compound. NE or saline wasadministered over 80 s in a final volume of 1 ml, followed by 400  A l of saline to flush the intravenous line. 2.5. Statistical analysis Time courses for LDF were exported from the Biopacsoftware at a time resolution of 10 s per data point. For eachanimal, the average LDF over a 50-s time window coveringthe peak response (1 min 20 s to 2 min postinjection) wasused as a summary statistic of the relative change in each parameter. LDF time courses were normalized to the meanof a 5-min baseline period prior to the challenge and, thus,expressed as a fractional change. Statistical analysis of LDFwas performed on the fractional changes relative to one.LDF values were compared between vehicle and NE-  A. Gozzi et al. / Magnetic Resonance Imaging 25 (2007) 826–833 828  challenged groups by a one-way ANOVA followed by aDunnett’s test for multiple comparisons.The arterial blood pressure (BP) probe was calibratedwith an external reference, and MABP values wereexpressed as millimeters of mercury. MABP time courseswere exported at a resolution of 10 s per data point, and theaverage MABP of six time points covering the peak response(1 min 54 s to 2 min 6 s postinjection) was used as summarystatistic. Preinjection baseline MABP values (averaged over 5 min preceding the injection) in the rCBVand LDF cohortswere similar (88.6 F 1.2 and 93.8 F 3.1 mmHg, respectively;  P  =.15). Since the profile and magnitude of the MABPresponse to NE were also equivalent in the two cohorts andno statistically significant difference was observed at anyof the NE doses tested (two-tailed Student’s  t   test,  P  z .52),MABP data were pooled together to simplify the descrip-tion of the results. MABP values were compared betweenvehicle and NE-challenged groups using a one-way ANOVAfollowed by a Dunnett’s test for multiple comparisons.Correlation coefficients for basal, pre- or post-  p a  CO 2 and LDF or rCBV were calculated by linear regressionanalysis, with a level of significance of .05.Toinvestigatetheintegrityofautoregulation, ascatter plot of all the individual time points ( n =2340) recorded duringthe simultaneous measurement of arterial BP and LDFwas created. Arterial BP values were binned on the basis of 10-mmHg subdivisions, to improve the clarity of the plot.Linear regression analysis (least-squares fit) was performedfor data within the autoregulation range (60–120 mmHg). 3. Results Intravenous administration of saline did not affect  baseline MABP values. NE (0.125, 0.5, 2 and 8  A g/ml) produced fast-onset dose-dependent rises in MABP(98.1 F 5.5, 115.7 F 4.3, 141.2 F 6.8 and 159.8 F 5.9 mmHgat peak, respectively; Fig. 1A). At the three highest doses, theeffect  reached statistical significance (  P  b .0001 vs. saline,Fig. 1B). The changes were abrupt and short-lived, withreturn to preinjection baseline values typically within 5 min. No significant changes in mean LDF were observed at the two lower doses of NE, despite peak MABP changes up Fig. 1. (A) Temporal profiles of MABP. NE or saline was injected at 0 min. (B) MABP group mean response to NE. (C) Time profile of LDF in the S1FLcortex. NE or saline was injected at 0 min. The horizontal line above the  x -axis indicates the infusion time. (D) Group mean response of LDF changes in themotor cortex (statistical significance of one-way ANOVA with Dunnett’s correction for multiple comparisons: *  P  b .05, **  P  b .01, ***  P  b .0001 vs. vehicle).The horizontal line above the  x -axis indicates the infusion time. The error bars of the temporal profiles have been omitted to improve the clarity of display.Fig. 2. Relative LDF versus BP (mean F 0.95 confidence intervals). The broken line shows the linear least-squares fit to the LDF data between60 and 120 mmHg.  A. Gozzi et al. / Magnetic Resonance Imaging 25 (2007) 826–833  829  to 115 mmHg (NE 0.5  A g/kg). Transient increases in meanLDF (+30.6 F 10.1% and +37.4 F 12% at peak, respectively;  P  b .05 vs. vehicle baseline, Fig. 1C and D) were observedwith larger doses of NE (2 and 8  A g/kg;  P  b .05 and  P  b .01,respectively), corresponding to MABP responses in excessof 120 mmHg.The correlation of LDF and arterial BP is shown inFig. 2. The small LDF reactivity (0.17% per mmHg,  P  =.00001) for BP values between 60 and 120 mmHgsuggests that autoregulation is preserved in this range. AsBP exceeded 120 mmHg, a clear breakdown in CBFautoregulation was observed, with LDF becoming highlydependent on BP (0.48% per mmHg). A similar trend wasalso noticeable with BP values below 60 mmHg.The microvascular rCBV response to NE was consistent with that of LDF. Saline injection induced a slight, transient signal decrease. This effect is probably the result of temporary dilution of the blood-pool intravascular contrast  Fig. 3. (A) Group mean response of microvascular rCBV from representative VOIs at different NE doses (statistical significance of one-way ANOVA withDunnett’s correction for multiple comparisons: *  P  b .05, **  P  b .01 vs. vehicle). (B) Temporal profiles of microvascular rCBV following NE challenge at different doses measured in the hippocampus and S1FL cortex. NE or saline were injected at 0 min.Fig. 4. Statistical parametric maps of the rCBV changes induced by increasing doses of NE versus vehicle, thresholded using clusters determined by  Z  N 2.3 anda (corrected) cluster significance threshold of   P  =.05. The figure also illustrates the region of insertion of the LDF probe within the S1FL cortex.  A. Gozzi et al. / Magnetic Resonance Imaging 25 (2007) 826–833 830
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