Proc. Natl. Acad. Sci. USAVol. 92, pp. 1846-1850, March 1995Medical Sciences

Quantitative measurement of regional blood flow with gadoliniumdiethylenetriaminepentaacetate bolus track NMR imaging incerebral infarcts in rats: Validation with theiodo[14C]antipyrine technique

(T2 NMR imaging/quantitative noninvasive regional cerebral blood flow measurement/autoradiography/cerebral focal ischemia)

FRANK WITrLICHt, KANEHISA KOHNOt, GUNTER MIESt, DAVID G. NORRISt, AND MATHIAS HOEHN-BERLAGEt§tMax-Planck-Institut for Neurological Research, Gleuelerstrasse 50, D-50931 Cologne, Federal Republic of Germany; and tUniversity of Bremen,Leobenerstrasse, D-28334 Bremen, Federal Republic of Germany

Communicated by Louis Sokoloft National Institutes of Health, Bethesda, MD, October 27, 1994 (received for review July 28, 1993)

ABSTRACT NMR bolus track measurements were corre-lated with autoradiographically determined regional cerebralblood flow (rCBF). The NMR method is based on bolusinfusion of the contrast agent gadolinium diethylenetriamine-pentaacetate and high-speed T *-sensitive NMR imaging. Thefirst pass ofthe contrast agent through the image plane causesa transient decrease of the signal intensity. This time courseof the signal intensity is transformed into relative concentra-tions of the contrast agent in each pixel. The mean transit timeand relative blood flow and volume are calculated from suchindicator dilution curves. We investigated whether this NMRtechnique correctly expresses the relative rCBF. The relativeblood flow data, calculated from NMR bolus track experi-ments, and the absolute values of iodo[t4C]antipyrine auto-radiography were compared. A linear relationship was ob-served, indicating the proportionality of the transient NMRsignal change with CBF. Excellent interindividual reproduc-ibility of calibration constants is observed (r = 0.963). For agiven NMR protocol, bolus track measurements calibratedwith autoradiography after the experiment allow determina-tion of absolute values for rCBF and regional blood volume.

To understand the evolution of cerebral lesions, informationon threshold conditions of cerebral blood flow (CBF) is oftenof importance (1, 2). Thus, absolute values of regional (r) CBFare needed. Radioactive tracer techniques have been devel-oped (3-5) to access data on quantitative blood flow. Thesemethods are invasive and do not permit study of the evolutionof pathophysiological changes. A noninvasive technique wouldpermit repetitive CBF measurements on individuals to studytemporal alterations of biochemical and metabolic processes inassociation with hemodynamic changes.

Belliveau et al. (6) and others (7, 8) have described atechnique to obtain regional cerebral blood volume (rCBV) byusing noninvasive T 2-sensitive NMR bolus track measurements,based on indicator dilution methods (9, 10). This techniquewas shown to be linearly proportional to changes of Pco2 (6,11), indicating information on relative rCBV changes underchanging physiological conditions. The approach, limited tothe determination of relative values of rCBV from NMR dataalone, has been shown to be sensitive to blood flow changes (7,8). This apparent sensitivity to CBF has been used for the studyof stroke patients (12), traditionally a domain of positron-emission tomography (13) and single-photon emission com-puter tomography (SPECT) (14). Yet, demonstration of adirect and linear relationship between the NMR-derivedrCBF-sensitive signal change and true rCBF values is stilllacking.

Here, we describe the correlation of the NMR T *-basedbolus track method with the invasive autoradiography ofiodo[14C]antipyrine (IAP) infusion (4). The rCBF values of theIAP method are used to calibrate the NMR data from thesame individuals. The correlation of rCBF, derived from NMRbolus track using the central volume principle (15), withautoradiographical rCBF values is assessed in a rat model offocal cerebral infarct providing a wide range of CBF values:contralateral, normal CBF, periinfarct (reduced) CBF, andlow CBF in the lesion center. The interindividual reproduc-ibility of the proportionality between autoradiography andNMR bolus track is tested by comparing data of an ensembleof animals under identical experimental conditions.

MATERIALS AND METHODSAnimal Model. Male Wistar rats [346-410 g (body weight)]

were anesthetized with 0.8% halothane in a 7:3 (vol/vol)N20/O2 mixture, tracheotomized, and mechanically venti-lated. Blood samples were obtained via an arterial catheter;two venous catheters were used for injection of pancuroniumbromide (0.5 mg per kg per h i.v.) and of gadolinium dieth-ylenetriaminepentaacetate (Gd-DTPA). Body temperaturewas recorded with a rectal thermometer and was kept at 37°Cwith a feedback-controlled warm-water blanket. The animalswere placed in a stereotaxic headholder for positioning in themagnet. Two graphite electrodes were fixed epidurally forrecording of electroencephalogram and dc potential duringthe NMR experiment. Focal ischemia was produced by in-traluminal occlusion of the middle cerebral artery (MCA), asdescribed (16). A 3-0 nylon thread, connected to a wire forguidance of a catheter, was inserted into the internal carotidartery through the mobilized proximal end of the externalcarotid artery. The arrangement allowed the manipulation ofthe thread position from outside the magnet with no need tomove the animals. Occlusion was monitored by decrease of theelectroencephalogram amplitude.NMR Experiments. The NMR experiments were performed

on a 200-MHz Biospec system (Bruker, Karlsruhe, Germany)with a 30-cm horizontal magnet. The system was equipped withan actively shielded gradient coil system (maximum gradient, 100mT/m; rise time < 250 ,us). Excitation and signal detection wasachieved with an Alderman-Grant resonator. For the perfusionmeasurements, a T *-sensitive version of the ultrafast imagingtechnique UFLARE (11) was used. Image matrix was 64 x 128with a relaxation time of 1000 ms, resulting in a recording time of

Abbreviations: Gd-DTPA, gadolinium diethylenetriaminepentaac-etate; CBF, cerebral blood flow; rCBF, regional CBF; rCBV, regionalcerebral blood volume; IAP, iodo[14C]antipyrine; MCA, middle ce-rebral artery; MTT, mean transit time; CBFi, index of CBF; ROI,

I region of interest.§To whom reprint requests should be addressed.

1846

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

Proc. Natl. Acad. ScL USA 92 (1995) 1847

1 s per image. Slice thickness was 2.5 mm; field of view was 4.5cm.

Bolus track experiments were performed in a coronal planeduring the control phase and 120 min after MCA occlusion.The plane position was chosen 5 mm posterior to the rhinalfissure to cover the central part of the infarcted territory. FiftyT*-sensitive images were recorded consecutively. Synchro-nously with the start of the 11th image, Gd-DTPA at 0.30mmol/kg (body weight), dissolved in 0.35 ml of Ringer'ssolution, was injected into the femoral vein over a 2-s interval.

Autoradiography. Directly after the bolus track NMR mea-surement after MCA occlusion, CBF was measured by intrave-nous ramp infusion ofIAP (40 ,uCi in 1 ml of physiological saline;Amersham; 1 Ci = 37 GBq) for 1 min while collecting arterialblood samples onto preweighted filter paper. At the end of thetracer infusion, the animals were immersed in liquid nitrogen.The brains were removed from the skull at -20°C and cut seriallyinto 20-,um-thick cryostat sections. Brain sections were exposedto x-ray film (Hyperfilm, Kodak) for 8 days, with calibrated14C-labeled polymer sheets. The resulting autoradiograms and14C standards were digitized with a charge-coupled device cam-era attached to an image processing system. Evaluation of localCBFwas performed after measuring the 14C in the sampled bloodwith a liquid scintillation counter (Wallac 1410, Pharmacia) (4).Alignment and calculation of the quantitative blood flow imageswere done on a Macintosh computer (Apple) by using IMAGEsoftware (National Institutes of Health, Bethesda).Computation of NMR Parameter Images and Image Anal-

ysis. The time course of the signal intensity was calculated foreach pixel. As described below, from this time course, the bloodflow parameters were obtained, leading to parameter images.The calculation is based on tracer dilution methods (9, 10).

First, signal intensities were transformed into relative con-centrations c(t) of the tracer (6):

1 s(t)c(t) I- n-so.TEk1 o 1

TE (in our experiments, 5 ms) is the interecho time, So is thebaseline signal intensity, s(t) is the time-dependent signalintensity, and k1 is a tissue-, pulse-sequence-, and field-strength-specific constant. The proportionality between c(t)and the signal intensity change had been shown with empiricaldata (7, 11, 17, 1) and Monte Carlo modeling calculations (18).Based on the central volume principle (15), the integral

under this concentration time course results in the determi-nation of CBV except for a scaling constant (see below):

c x 0

rCBV= fc(t)dt/fc*(t)dt. [2]o 0

The vascular transit time was calculated as the first moment ofthe fitted concentration time curve, corrected for the time ofarrival. The validity of the central volume principle for thedetermination of the mean transit time (MTT) from NMRbolus track data has been challenged (19, 20). These authorsclaimed that MTT being the first moment of the concentrationtime curve could only be seen as an approximation and that,for an accurate theoretical approach, an appropriate physio-logical model for tissue blood flow was needed (20). Becausesuch a model is not available, to our knowledge, we decided touse the first moment as a first approximation:

00 co

rMTT = ftc(t)dt/fc(t)dt, [3]o 0

where rMTT is the regional MTT. Consequently, by keepingin mind that the determination of MTT must be taken as anapproximation, an index of CBF (CBFi) was estimated (19)from the values of the determined CBV and MTT, based onthe central volume principle, where CBFi is the ratio CBV/MTT. c(t) is the time-dependent concentration in the tissue asdetermined in Eq. 1. The denominator of Eq. 2 is the timeintegral of the arterial bolus input function ct,(t), corrected forrecirculation of tracer. This arterial input function cannot bederived from the NMR data. This denominator of Eq. 2 is aglobal integral (i.e., has one global fixed value for an individ-ual) that is equal for all pixels in the image plane and may beseen as a proportionality constant. But, the value of this globalintegral depends on the particular physiological situation ofthe individual under investigation. Furthermore, this integralcan be expressed as the ratio of total quantity of tracerinjected, Qo, divided by the steady-state cardiac output KWe emphasize that an assumption of an individual-

independent value of this integral will not hold. It remains tobe assessed whether the assumption is valid for closely similarphysiological states of the animals.By inserting Eq. 1 into Eq. 2, we obtain Eq. 4 that correlates

the NMR signal intensity change with rCBV (and with rCBFby using Eq. 3 and the relation CBFi = CBV/MTT):

k' s(t)rCBV= TE ln -dt, [41TE [4

where k' summarizes both k, and the time integral of the arterialbolus input function ct. Both rCBV and rCBFi are only deter-mined relatively because the constant k' cannot be derived fromthe NMR data alone. For an absolute determination of CBFi,either the scaling factor k' must be determined or the NMR datamust be correlated with independent absolute CBF data.

Fit of the Concentration-Time Curve. The c(t) was calcu-lated from theNMR signal intensity change by using Eq. 1. Forthe time integral of the concentration-time curve, the exper-imental data points were fitted to a function. For best resultsin terms of accuracy, fitting stability, and minimal errors,various fitting algorithms were tried (21): For the analysis ofthe y variate function as described by Thompson et al. (22),either a least squares estimation based on the Gauss-Newtonmethod with a modification by Hartley (23) or the method ofthe moments was applied. Alternatively, the concentration-time curve was interpolated by using a cubic spline function(24). Finally, the Simpson and the trapezoidal procedures weretested for the calculation of the integral. Systematic analysis ofthe quality of these competing procedures showed the highestaccuracy and reliability for the Gauss-Newton least squaresestimation of the 'y variate parameters or the Simpson method(21). These two methods were, therefore, used for the pixel-wise calculation of the parameter images for CBV, CBFi, andMTT. All evaluations were carried out with FLOWIMAGEsoftware (21) on a Macintosh computer.

Regional Correlation ofAutoradiograms and NMR Images.A discrepancy between both imaging methods lies in theirdiffering slice thickness: 20 ,um for the autoradiographicsections and 2.5 mm for the NMR images. For a regionalcorrelation of both image types, partial volume effects must beminimized. Therefore, 8-10 autoradiograms spanning a vol-ume comparable to that in the NMR experiments were aver-aged. To this end, the individual sections were aligned inter-actively on the computer by using morphological landmarks.Then, a new blood flow image was calculated by pixelwise

lBelliveau, J. W., Kantor, H. L., Pykett, I. L., Rzedzian, R. R., Ber-liner, E., Beaulieu, P., Buonanno, F. S., Brady, T. J. & Rosen, B. R.,Society of Magnetic Resonance in Medicine, Aug. 20-26, 1988, SanFrancisco, p. 222 (abstr.).

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1848 Medical Sciences: Wittlich et al.

Table 1. Physiological parameters 2 h after occlusion of the MCA

Parameter Value

Arterial pH 7.36 ± 0.02Arterial Po2, mmHg 122.6 ± 16.8Arterial Pco2, mmHg 43.0 ± 3.9Hematocrit 45 ± 4Glucose, mg 151 ± 14MABP, mmHg 113 ± 7Body temperature, °C 36.6 ± 0.9

All values are mean ± SD (n = 5). MABP, mean arterial bloodpressure.

averaging the CBF values of the image stack. Comparison ofCBF values from both imaging methods was not possible on apixel-by-pixel basis. The autoradiograms have a high resolutiondue to the film used. In theNMR images, the spatial resolutionis compromised to record the signal intensity change duringthe bolus pass with high temporal resolution. Therefore, thepixel sizes in both pictures were related to the brain size. Theregion of interest (ROI), selected in one image, was scaled tothe same position and the same size in the other image, thusassuring same size and position of the ROIs in both images.

RESULTSGeneral Physiological Observations. Table 1 summarizes the

average physiological parameters measured 2 h after MCAocclusion, directly before the bolus track experiment. The phys-iological condition was maintained in the normal range and didnot change significantly during the whole observation period.NMR Bolus Track Measurements. Fig. 1 shows the sensitivity

of the UFLARE method to T 2 changes caused by the passage of thecontrast agent through the selected slice. The time course ofsignal intensities of three ROTs in the contralateral and ipsilateralsides and the border of the infarct are given. The signal intensitydecrease is 45% in normal brain, 25% in the border of the infarct,and <8% in its center. Fig. 2 presents six images at various timesduring the bolus track experiment after MCA occlusion of oneanimal. A transient signal decrease during the passage of thebolus is visible in the normal hemisphere, while little change insignal intensity is noted in the occluded part of the brain.

Calibration of the NMR Bolus Track Method. For calibra-tion, the values of the bolus-track-derived relative rCBFi dataare correlated with those rCBF values of the IAP method incorresponding ROTs (Fig. 3). To obtain a wide range of bloodflow values, ROTs are evaluated from normal tissue, from theinfarct core, and from various positions in the infarct borderwith a variable degree of reduction in blood flow (Fig. 3). Thiscorrelation resulted in an excellent linear relationship (r =

1.1

1.0

(.9r.

0.4-

CU

Co

0.8

0.7

0.6

0.50 10 20

Time, s

Proc. NatL Acad Sci USA 92 (1995)

FIG. 2. Six T *-sensitive coronal images from a series of 50 imagesof the bolus track experiment 2 h after MCA occlusion in a rat. Thetransient signal decrease is visible in the normal brain hemisphere(right sides of each image). Little change is observed in the ischemicpart of the brain (left sides of each image) because no contrast agentreaches it due to the MCA occlusion.

0.932), as shown in Fig. 4 for the ROTs marked in Fig. 3. Notethat the CBFi values from the NMR bolus track experimentsare given in arbitrary units because the scaling factor k' cannotbe derived from the NMR data analysis alone.The interindividual reproducibility of the method for quan-

tification of rCBFi with NMR bolus track is assessed bycomparing correlations of different animals under identicalexperimental conditions. Slope m and intercept b of the linearfit with the ordinate, obtained from the regression analysis, arelisted in Table 2 for five animals and show a very high degreeof reproducibility. Even when all rCBF data from all ROTs ofall animals are pooled, the regression analysis of the pooleddata still leads to a correlation coefficient r = 0.945, whileslope m (1.049) and intercept b (-17.7) of the fit show littlevariation from the values of individual animals (Table 2). Theparameters b and m, obtained from the ROI-based calibration,are used for pixel-value transformation to compute absolute-value rCBF bolus track images. MTT is calculated from theNMR data, in units of time. Thus, rCBV images can bequantified by using CBV = MTT x CBF.

Fig. 3 shows an autoradiographic presentation of rCBF and thecorresponding calibrated parameter images of rCBV, rCBF, and

30 40

FIG. 1. Signal intensities in re-

sponse to the bolus track experimentfrom three ROIs of a rat brain, 2 hafter MCA occlusion: contralateralnormal brain tissue (0), infarct core(+), and border zone of the infarct(*). The hatched area marks the timeof bolus injection.

: I. ta

s.': t :.:: + + At +** St*oto;E ;

r~ ~ ~ ~~~~~A_f * .*

F4Jt ~ ~ ~

5{

Proc. Natl. Acad. Sci. USA 92 (1995) 1849

FIG. 3. Coronal parameter im-ages of rCBV, rCBF, and rMTT,calculated from NMR data, afterthe calibration of the NMR-basedrCBF with the IAP technique. Forcomparison, the corresponding au-toradiogram is included as indi-cated. ROIs on both hemispheresare depicted in identical locationsof the autoradiogram and the cal-ibrated NMR-based CBF image.These ROIs were used for Fig. 4,indicating the linear correlationbetween both CBF imaging tech-niques.

rMTT from the NMR bolus track experiment. Good agreementof the ischemic region (i.e., the area of reduced blood flow)between the images from the bolus track measurement and theautoradiography is noted. The infarct areas, determined as thoseterritories with blood flow .25 ml per 100 g per min andexpressed relative to the area of the ipsilateral hemisphere, are 43+ 13% and 41 + 14% for the bolus track and the autoradio-graphic data, respectively. Thus, regional agreement betweenboth CBF imaging techniques is also good.

DISCUSSIONThe NMR imaging method UFLARE used in the present study hasa satisfactory temporal resolution for recording the first passageof the contrast agent. The sensitivity of the method for T 2variations is demonstrated for regions with differing blood flow.An excellent linear correlation of CBF values in corresponding

ROIs in both image types (i.e., NMR bolus track and autora-diography) was found. Thus, the NMR bolus track informationdirectly reflects relative rCBF changes. With the rat model offocal cerebral ischemia, we were able to show that this linearrelationship between NMR signal changes and absolute rCBFvalues, obtained from an independent method, holds over a widerange of CBF values. It is, therefore, correct to use the NMRbolus track signal intensity changes to express relative rCBF.

160

S. 120

.o X80

0

40u

0 40 80 120 160rCBF (NMR bolus track), AU

FIG. 4. Correlation of the blood flow data from measurements ofNMR bolus track and IAP for one animal. An excellent linear relation-ship is observed. The correlation factor r is 0.932. AU, arbitrary unit(s).

A very high interindividual reproducibility of the linear rela-tionship between NMR bolus track signal changes and autora-diographically obtained rCBF values was observed for animalsunder comparable physiological conditions and cardiac output.Thus, k' can in fact be used as a constant, provided the physio-logical conditions of the animals (and the imaging conditions)remain unchanged. Therefore, calibration of the NMR data maybe performed in only some of the group of animals used for aparticular investigation, under the precondition of a constantNMR measurement protocol and a constant infusion protocol.To strengthen the justification of such an extrapolation from afraction of calibrated data to uncalibrated data, femoral bloodsampling could be performed, parallel to the CBF measurement.This would give the cardiac output by dividing the dose of thecontrast agent, injected at zero time by the time integral of thefemoral arterial tracer concentration between zero and infinitetime.Combination of the NMR bolus track measurements and a

method for absolute determination of rCBF should allow thecalibration of the relative rCBF values of the NMR technique.Due to the determination of rMTT by the NMR method,regional blood volume can also be quantified. The exactdetermination of rMTT depends on the validity of the centralvolume principle (15) under the NMR protocol. Hamberg et al.(19) and Weisskoff et al. (20) have expressed reservationsabout the applicability of the central volume principle to NMRbolus track experiments. According to them, the expression ofrMTT as the first moment of the concentration-time curve ofNMR experiments is only an approximation with the truevalue of rMTT depending on the topology of the vessels. Thus,

Table 2. Correlation coefficient r, slope m, and intercept b withthe ordinate of the calibration curve: rCBF (NMR bolus track) vs.rCBF (IAP)

Animal r m b

1 0.991 0.961 -12.12 0.992 1.026 -12.03 0.996 1.027 -6.74 0.885 1.194 -31.75 0.949 1.064 -22.8

Average 0.963 ± 0.047 1.054 + 0.086 -17.1 + 10.1Pooled data 0.945 1.049 -17.7

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1850 Medical Sciences: Wittlich et al.

a precise determination of rMTT must await the availability ofan appropriate physiological model for tissue blood flow (20).

Potential over- or underestimation of the rMTT values willlead to a scaling error of the CBFi values extracted from theNMR data. As the NMR-based rCBFi data were calibrated byan independent method, the resultant absolute values are notaffected by such a scaling error in rMTT. Moreover, therelative rCBFi data entering our correlation with autoradiog-raphy data are derived from correct relative rCBV and abso-lute rMTT data. Thus, the observed linear relationship be-tween the rCBF data of both methods indicates the reasonableaccuracy of the mathematical derivation of rMTT. At any rate,errors in the calculation of rMTT are expected to be limitedto a linear proportionality constant.The quantitative absolute rCBV data are the product of the

calibrated rCBF data and the rMTT data. Comparison ofrCBV values from our evaluations (cortex, 5.17 ± 1.70%;striatum, 3.40 ± 0.76%) do not disagree substantially withliterature values: Shockley and LaManna (25) reported 3.4%under normoxic normocapnic conditions; White et al. 1 noted2.4 ± 0.3% for hemispheric values; Weiss et al. (26) found 2.85+ 1.44%. In experiments of focal rat brain ischemia, Kent etal. (27) described CBV values of the contralateral normalhemisphere of 0.9-5.2%. Thus, the calculation errors of ourrCBV calibration, introduced by the simplified assumptionabout the rMTT determination, appear only minor.The fact that the calibration curve (Fig. 4) shows an intercept

not equal to zero indicates a systematic error. The followingpotential reasons must be discussed. The accuracy limits of thefitting procedures to the concentration time curve (due to thetemporal resolution and the limited signal-to-noise ratio in theexperimental data) could result in an overestimation of the CBVvalues. This would explain that the herein calculated CBV is onthe high end of the range described in the literature (see above).At the same time, too high values for MTT would lead toartificially low CBFi values (=CBV/MTT), thus, resulting in anintercept not equal to zero in the calibration curve. Thesepotential errors will, however, not affect the results of a linearrelationship between the bolus-track-induced signal intensitychange and the independently determined rCBF.Other described NMR imaging methods for the determina-

tion of CBV (28, 29) depend on T1 relaxation changes afterinfusion of intravascular contrast agents. These methods de-termine only CBV, not CBF or MTT, and depend on bloodsampling or simultaneous recording of signals arising solelyfrom blood protons like the sagittal sinus. Furthermore, floweffects are not included in the signal dependence from suchregions of interest. Rudin and Sauter (30) have described thecalibration of NMR signal intensity changes after a 2-minGd-DTPA infusion with IAP autoradiography. These authorsused a long-lasting tracer infusion so that no transient signalintensity change was measurable by NMR. Instead, theyobserved only the wash-out curve of the tracer. Therefore,determination ofMTT was precluded from their experimentaldata, and the analysis had to be restricted to a comparison ofthis wash-out behavior with independently measured CBFdata. Consequently, rCBV or rMTT could not be described.We emphasize here that the noninvasiveness of the NMR

technique allows repetitive measurements in an individual.The contrast agent is cleared from the blood stream (30)within 30 min of application, so that a quantitative measure-ment in the same animal is possible on an hourly basis. Thisopens possibilities for investigations concerning hemodynamic

dependencies of temporal evolution of pathophysiologicalprocesses. Pathophysiological models, like the one presented(brain infarct), can be studied with respect to changes ofquantitative rCBF and rCBV.

Thus, for a given experimentalNMR bolus track protocol, dataare obtained directly proportional to rCBF values over a widerange of absolute values. NMR bolus track signal intensitychanges correctly express relative rCBF. Calibration with anestablished regional technique forrCBF determination allows theabsolute determination of CBF and CBV from NMR data.

We thank Mrs. A. Gottschalk and Mrs. C. Stratmann for their experttechnical assistance with autoradiography. Stimulating discussionswith Prof. K.-A. Hossmann and Dr. M. L. Gyngell are gratefullyacknowledged. The project was supported by the Deutsche For-schungsgemeinschaft (SFB 194/Bl).

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