effect of hypoxia/hypercapnia on metabolism of 6-[18f]fluoro-l-dopa in newborn piglets

Brain Research 934 (2002) 23–33www.elsevier.com/ locate /bres

Research report18Effect of hypoxia /hypercapnia on metabolism of 6-[ F]fluoro-L-DOPA

in newborn pigletsa , b a b b*Reinhard Bauer , Peter Brust , Bernd Walter , Gerd Vorwieger , Ralf Bergmann ,

a a b b b¨ ¨Elsayed Elhalag , Anne Fritz , Jorg Steinbach , Frank Fuchtner , Rainer Hinz ,a aUlrich Zwiener , Bernd Johannsen

aInstitute of Pathophysiology, Friedrich Schiller University, D-07740 Jena, GermanybInstitute of Bioinorganic and Radiopharmaceutic Chemistry, Research Center Rossendorf, Germany

Accepted 12 November 2001

Abstract

There is evidence that the dopaminergic system is sensitive to altered p in the immature brain. However, the respective enzymeO2 18activities have not been measured in the living neonatal brain together with brain oxidative metabolism. Therefore F-labelled6-fluoro-L-3,4-dihydroxyphenylalanine (FDOPA) together with positron emission tomography (PET) was used to estimate the activity ofthe aromatic amino acid decarboxylase (AADC) in the brain of fifteen newborn piglets (2–5 days old). Two PET scans were performed ineach piglet. Eleven animals underwent a period of normoxia and moderate hypoxia /hypercapnia (H/H). The remaining four animals wereused as an untreated control group. Simultaneously, the brain tissue p was recorded, the regional cerebral blood flow (CBF) wasO2

measured with colored microspheres and the cerebral metabolic rate of oxygen (CMRO ) was determined. In addition, in four untreated2

and six H/H treated piglets the relative amounts of fluorodopamine and the respective metabolites were determined in brain tissuesamples using HPLC analysis. H/H conditions were induced by lowering the inspired fraction of oxygen from 0.35 to 0.10 and addingCO to the inspired gas resulting in an arterial p between 74 and 79 mmHg. H/H elicited a more than 3-fold increase of the CBF2 CO2

(P,0.05) so that the CMRO remained unchanged throughout the H/H period. Despite this, the brain tissue p was reduced from 19642 O2FDOPAto 663 mmHg (P,0.05). The permeability–surface area product of FDOPA (PS ) was unchanged. However, the transfer rate ofFDOPAFDOPA (k ) of the nigrostriatal dopaminergic system and the relative amounts of fluorodopamine and the respective metabolites were3

significantly increased (P,0.05). It is suggested that H/H induces an increase of AADC activity. However, an H/H-induced CBFincrease maintains bulk O delivery and preserves CMRO . 2002 Elsevier Science B.V. All rights reserved.2 2

Theme: Neurotransmitters, modulators, transporters and receptors

Topic: Catecholamines

Keywords: FDOPA; Aromatic amino acid decarboxylase; Dopamine metabolism; Positron emission tomography; Colored microsphere; Newborn piglet

1. Introduction of the chronic handicapping conditions of cerebral palsy,mental retardation, and epilepsy [49]. However, the imma-

In the perinatal period, infants with various clinical ture brain is considered to be relatively resistant todisorders are commonly exposed to periods of hypoxia / neuropathological damages from moderate H/H. Neverthe-hypercapnia (H/H). Severe H/H combined with brain less, exposure to moderate oxygen deficiency during earlyischemia, mostly due to secondary hypotension [50] and postnatal life, which does not cause neuronal damage, mayassociated with neuronal damage remains a frequent cause induce long-term alterations of the dopaminergic system. It

was shown that moderate hypoxia during early postnatallife may disrupt functional activity at selected synapses*Corresponding author. Tel.: 149-3641-938-956; fax: 149-3641-938-[26,27]. Furthermore, there is evidence that oxygen de-954.

E-mail address: [email protected] (R. Bauer). ficiency at critical stages in development may cause

0006-8993/02/$ – see front matter 2002 Elsevier Science B.V. All rights reserved.PI I : S0006-8993( 02 )02315-6

24 R. Bauer et al. / Brain Research 934 (2002) 23 –33

permanent changes in the dopaminergic synaptic function in nitrous oxide–oxygen (70:30, v /v) by a mask. The[11]. anesthesia was maintained throughout the surgical pro-

Sensitivity of the dopaminergic system in the immature cedure with 0.8% isoflurane. A central venous catheter wasbrain to oxygen deficiency may be related to alterations in introduced through the left external jugular vein and wasbrain tissue p . This was postulated because of a dose– used for the administration of drugs and for volumeO2 21substitution (lactated Ringer’s solution: 5 ml kg b.w.response relationship between the brain tissue p and theO2 21h , i.v.). An endotracheal tube was inserted through astriatal extracellular dopamine (DA) concentration [DA]e

tracheotomy. After immobilization with pancuronium bro-in newborn piglets [41] and a gradual striatal [DA]e21 21mide (0.2 mg kg b.w. h , i.v.), the piglets wereincrease in relation to the exposed O atmosphere [38].2

artificially ventilated (Servo Ventilator 900C, Siemens-Increased [DA] is known to be associated with pro-e

Elema, Sweden). The artificial ventilation was adjusted tonounced neuronal injury due to the hypoxic–ischemicperform normoxic and normocapnic blood gas values. Thebrain [22]. However, there is evidence that even moderateventilation was monitored by end-tidal carbon dioxidelevels of oxygen deprivation combined with an acutelyrecording (carbon dioxide Analyzer 930, Siemens-Elema).increased [DA] elicited regionally selective, long-terme

Polyurethane catheters (0.5 mm, I.D.) were advancedalterations in the catecholamine levels and turnover [45],through both umbilical arteries into the abdominal aorta inwhich is presumably based on persistent alterations in geneorder to record arterial blood pressure and to withdrawexpression during the critical developmental period. Thereference samples for the colored microsphere technique.extracellular levels of DA, however, do not necessarilyA further polyurethane catheter (0.3 mm, I.D.) was insertedrepresent the function of the neurons, since they depend oninto the superior sagittal sinus through a midline burr holeseveral factors: [DA] is determined by release, reuptake,e

(diameter 3 mm, 4 mm caudal to bregma) and advanced towashout from the brain and metabolism by enzymes suchthe confluence of the sinuses in order to obtain brainas monoamine oxidase (MAO) and catechol-O-methyl-venous blood samples. The left ventricle was cannulizedtransferase (COMT) [33]. The reason for the increasedretrogradely via the right common carotid artery with a[DA] due to oxygen deficiency is controversially dis-e

polyurethane catheter (0.5 mm, I.D.). Arterial, left ven-cussed. From in vivo studies on newborn rats it wastricular, and central venous catheters were connected withpostulated that hypoxia may increase the releasable dopa-pressure transducers (P23Db, Statham Instruments Hatomine pool [24]. However, other data suggested that a mildRey, Puerto Rico). The correct positioning of the catheterhypoxia-induced [DA] increase resulted from inhibition ofe

tips was checked by continuous pressure trace recordingsthe dopamine reuptake [2].and by autopsy at the end of the experiment. The bodyUntil now the effect of moderate H/H on the regionaltemperature was monitored by a rectal temperature probebrain dopamine metabolism in relation to the cerebraland was maintained throughout the experiment atoxidative metabolism has not been determined. Therefore,3860.3 8C using a warmed pad and a feedback controlledwe estimated the activity of the aromatic amino acidheating lamp. A hole was drilled into the left frontal bonedecarboxylase (AADC), the ultimate enzyme in DA syn-(2 mm in diameter) to implant a Clark-type p electrodethesis, together with the regional CBF, the brain tissue p OO 22

3–5 mm into the brain cortex together with a thermocoupleand CMRO and determined the relative amounts of2

catheter, serving as a temperature probe (Licox pfluorodopamine and the respective metabolites in brain O2

monitor, GMS, Kiel-Mielkendorf, Germany). All burrtissue samples using HPLC analysis under normoxicholes were sealed with bone wax and dental acrylic resin.conditions and during moderate H/H. We asked if H/H isPhysiological parameters were recorded on a multichannelable to induce alterations of the synthetic activity of thepolygraph (Gould, USA). The arterial blood pressure wasdopaminergic system in newborn piglets.monitored continuously, and arterial blood samples werewithdrawn and analyzed at regular intervals to monitor theblood gases and the whole acid–base parameters.2. Materials and methods

2.2. Experimental protocolAll surgical and experimental procedures were approvedby the committee of the Saxon State Government for

After the surgical preparation had been completed, theAnimal Research. The animals were managed in accord-anesthesia was reduced to 0.25% isoflurane in nitrousance with the guidelines of the American Physiologicaloxide–oxygen (65:35) and the piglets were allowed toSociety. Fifteen male newborn piglets of mixed Germanstabilize for 1 h. The piglets were studied lying prone in adomestic breed (aged 2–5 days, body weight 21406395 g)positron emission tomography (PET) scanner with thewere used in the study.head in a custom-made head-holder. The position of thehead was checked throughout the experiment with laser2.1. Surgical proceduresmarkers. Animals were randomized by lot and subsequent-ly subdivided into two groups. Four animals were heldPiglets were initially anesthetized with 1.5% isoflurane

R. Bauer et al. / Brain Research 934 (2002) 23 –33 25

under unchanged conditions throughout the experiment and by a diode-array UV–vis spectrophotometer (Model 7500,served as sham-operated control (group 1). Eleven ani- Beckman Instruments, Fullerton, CA, USA). Calculations

mals, in the second part of the experiment, underwent a were performed using the MISS software (Triton Technol-change in their inspired gas composition (inspired fraction ogy). The number of microspheres was calculated usingof oxygen was lowered from 0.35 to 0.10 in exchange for the specific absorbance value of the different dyes. Allnitrogen and CO was added resulting in an arterial p reference and tissue samples contained .400 micro-2 CO2

spheres. For the whole brain a percent recovery ofbetween 74 and 79 mmHg) and served as the hypoxia /3.1261.28 (normocapnic normoxia) and 9.5963.45% (hy-hypercapnia (H/H) group (group 2). The first PET studypercapnic hypoxia) of the totally injected microspheres waswas done under normoxic /normocapnic conditions in bothobtained.groups; 8 h later a second PET study was performed under

The heart rate, the arterial blood pressure, the arterialnormoxic /normocapnic conditions (group 1) or duringand brain venous pH, p and p , oxygen saturation andhypoxic /hypercapnic conditions (group 2). In group 2 the CO O2 2

the hemoglobin values were measured immediately beforesecond PET study was started 20 min after the beginningthe microsphere injection. The blood pH, p and pof hypoxia /hypercapnia. CO O2 218[ F]FDOPA was infused into the lower caval vein at a were measured with a blood gas analyzer (model ABL50,

rate of 10 ml /min. Emission scanning began simultaneous- Radiometer, Copenhagen, Denmark), the blood hemoglo-18ly with the start of [ F]FDOPA infusion (more details see bin and the oxygen saturation were measured using a

PET studies). CBF and CMRO were measured before hemoximeter (model OSM2, Radiometer), and the blood2

(Control 1) and 60 min after the first radiotracer infusion glucose and lactate contents were measured with an(Normoxia), 10 min before hypoxia /hypercapnia was electrolyte, metabolite laboratory EML105 (Radiometer)

introduced (Control 2), and 10 and 80 min after the and corrected to the body temperature of the animal at thebeginning of hypoxia /hypercapnia (group 2) and at corre- time of sampling.sponding times before and after the second PET study The absolute flows to the tissues measured by theduring normoxia (group 1), respectively. Blood volume colored microspheres were calculated by the formula:replacement was performed after every blood sampling flow 5number of microspheres ?(flow /tissue tissue reference

using stored heparinized blood obtained from a donor number of microspheres ). The flows are expressedreference

sibling piglet. In order to prevent a critical decrease of in milliliters per min per 100 g tissue by normalizing forarterial pH (,6.80) between 90th and 120th min of H/H, tissue weight. The cerebrovascular resistance (CVR) wasfour piglets received 2–5 ml sodium bicarbonate intraven- calculated as the quotient of the mean arterial bloodously. After the experiments were completed, the anes- pressure and the global brain blood flow (CBF). Assumingthetized animals were sacrificed by intravenous administra- the oxygen capacity of hemoglobin to be 1.39 ml O /g2

tion of saturated KCl solution. hemoglobin in piglets [5], the blood O content was2

calculated as equal to g hemoglobin /ml?1.39 ml O /g2

hemoglobin?%O saturation and expressed in mL/100 g2

2.3. Analytical procedures and calculations min. The CMRO was obtained by multiplying the blood2

flow to the forebrain by the cerebral arteriovenous O2

The regional CBF was measured by means of the content difference, where the blood flow to the forebrainreference sample color-labeled microsphere (Dye-Trak , includes all regions drained by the sagittal sinus (cerebral

Triton Technology, San Diego, CA, USA) technique, cortex, cerebral white matter, some deep gray structures:which represents a valid alternative to the radioactively basal ganglia, thalamus, and hippocampus) [14].labeled microsphere method for organ blood flow measure-ment in newborn piglets and avoids all disadvantagesarising from radioactive labeling [53]. Application in 2.4. PET studiespiglets and methodical considerations have been presented

18and discussed in detail elsewhere [6,53]. Briefly, in [ F]FDOPA was produced according to the destannyla-random sequence between 900 thousand and 1.2 million tion method by direct fluorination of the tin-precursor with

18colored polystyrene microspheres were injected into the [ F]F [39] simplifying the procedure (see [10]). The2

left ventricle. A blood sample was withdrawn from the piglets were studied lying prone in the scanner (Positomethoracic aorta as the reference sample. At the end of each IIIp (Montreal Neurological Institute); dynamic scans: 35experiment, the piglets’ brains were obtained. In order to frames between 30 and 600 s each, total length 120 min).

18retain the microspheres, each tissue sample was digested In each case 50–150 MBq [ F]FDOPA (in 10 ml) wereand then filtered under vacuum suction through an 8-mm infused within 60 s into the upper caval vein, followedpore polyester membrane filter. Colored microspheres were immediately by heparinized isotonic saline to flush thequantified by their dye content. The dye was recovered catheter. Fifty-two arterial blood samples in intervalsfrom the microspheres by adding dimethylformamide. The between 15 s and 30 min were obtained, stored on ice andphotometric absorption of each dye solution was measured centrifuged for plasma sampling. Plasma activity (100 ml)


was measured in a well counter (Cobra II) cross-calibrated duced by peripheral COMT at an apparent rate constantFDOPAwith the tomograph. Additionally nine blood samples (at 2, k are reversibly transferred across the blood–brain0

4, 8, 12, 16, 25, 50, 90 and 120 min) were withdrawn for barrier (BBB), a process described by the rate constantsFDOPA FDOPA OMFD OMFDHPLC analysis to correct the plasma input function for the K and k for FDOPA and K and k for1 2 1 2

18presence of [ F]FDOPA metabolites [51]. PET image data OMFD. Because there is a clear species dependency of theOMFD FDOPAwere reconstructed using a Hanning filter with a cut-off K :K ratio [18,52] data obtained in humans1 1

OMFDfrequency of 0.5. The spatial resolution was spatial res- could not be used. Therefore we have estimated K 1OMFDolution (transaxial) of 11 mm, a full width at half and k of newborn pigs in separate experiments. For2

maximum (FWHM) of 15 mm and an axial field of view both FDOPA and OMFD we have estimated the regionalFDOPA OMFDof 5 cm. Transmission scans were performed using three permeability–surface area products (PS and PS )

68rotating germanium ( Ge) sources to correct for attenua- of the endothelium and the regional equilibrium distribu-FDOPA OMFDtion. Regions of interest were set by hand according to tion volume (V and V ).e e

piglet brain stereotaxic coordinates [44]. In the brain OMFD is not assumed to participate in anybiochemical reaction, whereas FDOPA is decarboxylated

2.5. Model description and data analysis to fluorodopamine (FDA) by AADC at the rate constantFDOPAk . FDOPA is also a substrate for the COMT in the3

FDOPAThe compartment model which was applied to analyze brain (k ) but this is not considered in the model as518the transport and metabolism of [ F]FDOPA in the brain proposed also by others [30]. FDA is stored in vesicles or

is shown in Fig. 1. It is based on FDOPA models described further metabolized by the enzymes MAO and COMTin humans [30]. Briefly, the distribution of radioactivity yielding the acidic substances fluorodihydroxyphenylaceticoccurs between the intravascular space, the extravascular acid (FDOPAC) and fluorohomovanillic acid (FHVA). Inprecursor pool and the metabolite compartment and is addition, the rate of the conversion from FDA to fluoro-3-described by nine transfer coefficients. FDOPA and its methoxytyramine (3-FMT) by COMT has to be consid-methylated product 3-O-methyl-FDOPA (OMFD) pro- ered. These processes may be combined into a single

FDA1acidscompartment with the apparent rate constant kcl

which accounts for the clearance of labeled metabolitesfrom tissue.

18According to this model the total accumulation of F inthe brain regions, M(t), can be described by

M(t) 5

FDOPA FDOPA FDOPA FDOPAK C (t) ? exp [2(k 1 k )t]1 a 2 3

FDOPA FDOPA FDOPA FDOPA1 K k C (t) ? exp [2(k1 3 a 2

FDOPA FDA1acids1 k )t] ? exp (2k t)3 cl

OMFD OMFD OMFD1 K C (t) ? exp (2k t) 1V C (t) (1)1 a 2 0 a

FDOPA OMFDwhere C (t), C (t) and C (t) are the plasmaa a a18concentrations of FDOPA, OMFD and [ F]. V C (t) is the0 a

18amount of [ F] remaining in the brain vasculature.The relevant differential equations that describe the

changes of radioactivity contents in the compartments are:

OMFD OMFD OMFD OMFD OMFDdM /dt 5 K C (t) 2 k M (t) (2)f 1 a 2 fFig. 1. Compartmental model used to estimate the changes of AADC

FDOPA FDOPA FDOPA FDOPA FDOPAactivity during normoxia and H/H. The amino acids FDOPA and OMFD dM /dt 5 K C (t) 2 k M (t)f 1 a 2 fare reversibly transferred across the BBB. The transport is described byFDOPA FDOPA OMFD OMFD FDOPA FDOPAthe constants K and k for FDOPA and K and k for1 2 1 2 2 k M (t) (3)3 f

OMFD. In the brain tissue OMFD does not participate in any biochemicalreaction whereas FDOPA is decarboxylated by AADC at the rate constant FDOPA FDOPA FDOPA FDA1acids FDOPA

FDOPA FDOPA dM /dt 5 k M (t) 2 k M (t)k . It is also a substrate for the COMT in the brain (k ). The m 3 f cl m3 5

contribution of this process to the total OMFD activity in the brain is (4)negligible. The decarboxylation product fluorodopamine is stored in

OMFD FDOPAvesicles or further metabolized by the enzymes monoamine oxidase and where M (t) and M (t) are the amounts of freef fCOMT yielding the acidic substances fluorodihydroxyphenylacetic acid FDOPAOMFD and FDOPA in the brain. M (t) represents them(FDOPAC) and fluorohomovanillic acid (FHVA) which are eliminatedregional content of FDOPA metabolites.from brain. These processes are combined into a single compartment with

FDA1acidsthe apparent rate constant k . The rate constants for blood–brain and brain–bloodcl


FDOPAtransfer (K and k ), the apparent AADC activity (k ), volume 360 ml), UV detector (l5280 nm), all parts of the1 2 3FDA1acidsand the clearance rate constant (k ) were estimated Hewlett-Packard 1050 system, and a flow scintillationcl

by solving these differential equations for the measurable analyzer (150 TR, Canberra Packard) with a PET flow cellFDOPAvariables. As stated by Cumming and Gjedde [16] k (100-ml volume; energy window: 400–1500 keV). The3

is the fractional rate constant for decarboxylation, defined analytes were separated on a C reversed-phase column18

relative to the enzyme’s Michaelis constants (V /(K 1 (25034 mm, LiChrosorb, 7 mm) fitted with a guardmax m

[C]), where [C] is the concentration of L-DOPA, the column (434 mm, LiChrospher 100 RP-18, 5 mm), at aendogenous substrate, which is much less than K [16]. temperature of 30 8C. A binary gradient was chosen at am

The PS product was calculated from K and cerebral blood flow-rate of 1.5 ml /min (start: 0% B, 4 min: 25% B, 101

flow (F ) min 80% B, 10.1 min 100% B, 11.2 min: 100% B, 12.8min 0% B, 16 min: 0% B; total method time: 16 min).

PS 5 2 F ln (1 2 K /F ) (5)1 Mobile phase A consisted of 70 mM NaH PO , 10 mM2 4

sodium hexanesulfonate and 0.1 mM EDTA, adjusted toOMFD OMFDK and k were estimated from Eq. (2) in separate1 2 pH 3.42 with H PO . Mobile phase B was prepared by3 4experiments with OMFD as radiotracer. In FDOPA experi- adding two parts (v /v) acetonitrile (MeCN) to one part A.18F FDOPA OMFDments C , C and C were determined at ninea a a

time points from HPLC data. The data were fitted to an2.7. Statistical analysisempirical function whose parameters were later used to

FDOPAobtain a complete input function for C (t) andaOMFD OMFD Data are reported as means6S.D. Two-way analysis ofC (t) from C (t). The contribution of OMFD (M )a a f

variance (ANOVA), with repeated measures, was used toto the total brain activity was calculated from the in-OMFD OMFD determine the effects of the experimental procedure anddividual C (t) and the mean values of K anda 1

OMFD the protocol within physiological variables, cerebral bloodk for the various brain regions according to Eq. (2).2OMFD flows and parameters of brain oxidative metabolism andFor each time frame of the PET scans M wasf

between both groups. Posthoc comparisons were madesubtracted from M(t). Thereby the number of unknownwith the Tukey’s test for all pairwise multiple compari-parameters in Eq. (1) was reduced to five. They were

FDOPA sons. Comparisons between groups were made with un-obtained by least-squares fitting using C (t) and C (t)a apaired t tests using Bonferroni correction for multiple useas input functions. The rate constants for blood–brain andif indicated. One-way ANOVA was used to compare PETbrain–blood transfer (K and k ), the apparent AADC1 2

FDOPA FDA1acids data of different brain regions. Comparison of PET dataactivity (k ), the clearance rate constant (k )3 clbetween the two PET scan measurements was performedand the blood volume V were estimated from a double0using the paired t test. Differences were consideredintegral form of this compartmental model (analogous tosignificant when P,0.05.[8]).

2.6. Tissue sample preparation and HPLC analysis3. Results

18The metabolism of [ F]FDOPA in the piglet brain wasTable 1 summarizes the values for the mean arterialstudied by HPLC analysis [51]. Four control animals and

blood pressure (MAP), the heart rate, the arterial bloodsix piglets who underwent hypoxia /hypercapnia as de-gases, the acid–base balance and the energy fuels, whichscribed above were euthanized 120 min after injection of

18 were obtained during the blood flow measurements under[ F]FDOPA. The brain was rapidly exposed, dissected andnormoxic and H/H conditions. They cover a time schedulemechanically disintegrated. Samples from mesencephalon,of more than 8 h. The values measured under normoxicfrontal cortex and striatum were obtained. Each brainconditions before (control 1, time: 0) and during the firsttissue sample was immediately submerged in 2.5 ml ofPET-study (normoxia, time: 170 min) and before H/HCCl (precooled to 220 8C). 1 volume part (referring to4

onset (control 2, time: 18 h) were within the physiologicalthe obtained tissue weight) of a deproteinization solutionrange in both groups studied and consistent with other datacontaining 2.4 mol / l perchlorate and 0.8 mol / l Na HPO2 4

obtained from mildly anesthetized and artificially venti-were added and the sample was homogenized with anlated newborn piglets [20]. Widely unchanged valuesUltra Turrax. Then, another 1 volume part of 1 M KH PO2 4

during the first PET scan measurement and the restingsolution (referring to the tissue weight) was added. Afterperiod of about 8 h were also measured for the regionalbrief shaking the mixture was separated by centrifugationcerebral hemodynamics and the parameters of the cerebral(7 min at 16 000 g and 0 8C) into two solid (pellet andoxidative metabolism (Fig. 2). The brain tissue p ex-potassium perchlorate) and two liquid phases (CCl and a O4 2

clear supernatant). The supernatant was used for HPLC hibited a moderate decrement between the first and theanalysis using the following configuration: quarternary second period of PET measurements (control 1 vs. controlgradient pump, autosampler (0.5-ml sample loop; injection 2) which was more pronounced in group 1 (to 7364%,


Table 1Physiological values for newborn piglets during normoxia and hypoxia /hypercapnia

Group Control 1 1st PET Control 2 2nd PET 1 2nd PET 2

Mean arterial blood pressure 1 76611 7869 74615 79612 84617(mmHg) 2 75612 79612 69613 81613 75613

Heart rate 1 233639 242640 250635 239639 24064621 §(min ) 2 232635 242653 244640 269650 286665*

Arterial p 1 3863 3862 3862 3662 3861CO2 § §(mmHg) 2 3863 3862 3863 7964* 7464*

Arterial pH 1 7.4760.04 7.4660.04 7.4760.04 7.4860.03 7.4660.05§ §2 7.4560.04 7.4860.03 7.4760.04 7.1460.05* 6.9960.13*

Arterial base excess 1 461 462 362 361 36321 § §(mmol l ) 2 262 462 462 2363* 21366*

Arterial p 1 124625 119619 127633 122632 131643O2

(mmHg) 2 128621 122627 119621 3564* 4167*

Arterial oxygen content 1 5.360.3 5.160.4 5.060.5 5.460.3 5.460.721 § §(mmol l ) 2 4.960.8 4.760.9 4.760.9 1.660.2* 1.660.2*

Arterial glucose content 1 8.462.0 8.261.7 8.861.6 8.661.8 10.464.221(mmol l ) 2 7.060.8 7.361.0 7.461.4 10.062.6 7.762.9

Arterial lactate content 1 2.260.6 2.060.4 1.561.1 1.660.1 2.360.421 § §(mmol l ) 2 2.861.5 2.461.3 2.161.0 5.261.6* 13.565.1*

Av D-lactate 1 0.1460.29 0.1060.20 0.0060.09 0.0260.20 20.0960.74brain21 §(mmol l ) 2 20.0660.38 20.0460.23 20.1060.26 0.0560.54 0.5761.10

Values are means6S.D.; group 1: n54, group 2: n511; before (control 1) and during 1st PET scan procedure (60 min after FDOPA injection) and before(control 2) and during the 2nd PET scan procedure (2nd PET 1 indicate 10 min before; 2nd PET 2 indicate 60 min after FDOPA injection), av D-lactate:brain

brain arteriovenous difference of lactate content.§ §* , P,0.05; *, significant differences between group 1 and group 2, , significant differences within the related group compared with Control 1).

FDOPA FDOPAsignificantly reduced in comparison to control 1, P,0.05) During moderate H/H K and k tended to1 2FDOPAcompared to group 2 (to 88622%) (Fig. 2). increase in all brain regions and was significant for k2

18Regional transport of [ F]FDOPA to the brain indicated in frontal cortex and striatum (Table 2, P,0.05). The PSFDOPA FDOPA FDOPAby K and PS was quite similar during normoxic product of FDOPA (PS ) was unchanged. Further-1

18conditions (Table 2) between both groups. In addition, more, the rate constant for [ F]FDOPA decarboxylationFDOPAthere were no significant differences between the various (k ) was markedly increased in mesencephalon (41%)3

brain regions concerning the rate constants for backflux and striatum (32%) during H/H (Table 2; P,0.05)FDOPA FDOPAfrom the brain (k ) and V . The decarboxylation indicating a distinct increase of AADC activity during2 e

18 FDOPA FDA1acidsof [ F]FDOPA indicated by the rate constant k H/H. The clearance rate constant k was not3 cl

exhibited marked interindividual variance. The clearance changed under conditions of moderate H/H. In addition,FDA1acidsrate constant k was similar in all brain regions. the percentage of FDA, 3-FMT, FDOPAC and FHVA wascl

The supposed degree of moderate H/H, i.e. a reduction significantly increased, which is consistent with a higherof p of about one third of baseline value in addition to dopamine turnover caused by an increase of the AADCO2

nearly doubling p , led to a significant increase of heart activity (P,0.05, Fig. 3).CO2

rate (P,0.05) at maintained MAP together with combinedrespiratory and metabolic acidosis and progressively in-creased plasma lactate content (P,0.05). Furthermore, a

4. Discussioncorresponding decrease in arterial oxygen content to aboutone third occurred (P,0.05). Brain tissue p was mark-O2

In this study we have shown that moderate H/H inducededly reduced as well (Fig. 2, P,0.05). However, underan increase of the AADC activity within the mesence-this amount of H/H brain oxidative metabolism hasphalon and the mesotelencephalic dopaminergic systemobviously not been compromised. A pronounced CBF(striatum and frontal cortex). Nearly all animals studiedincrease (P,0.05) and a considerable CVR reduction

FDOPAshowed consistently an increase of k in the nigros-(P,0.05) resulted in an unchanged CMRO (Fig. 2). 32

triatal regions. Furthermore, the HPLC analysis has shownFurthermore, during the late period of moderate hypercap-that the relative amounts of fluorodopamine of mesence-nic hypoxia an increased cerebral arteriovenous differencephalon and frontal cortex and the derived acidic metabo-of lactate occurred (Table 1, P,0.05).


Fig. 2. Cerebral metabolic rate of oxygen (CMRO ), cerebral blood flow (CBF), cerebrovascular resistance, and brain tissue p in normoxic control2 O2

(group 1: n54, filled columns) newborn piglets and piglets exposed a period of moderate hypoxia /hypercapnia (H/H) (group 2: n511, open columns; 2ndPET 1 (10th min H/H), 2nd PET 2 (80th min H/H)) before (control 1) and during 1st PET scan procedure (60 min after FDOPA injection) and before(control 2) and during the 2nd PET scan procedure (2nd PET 1 indicates 10 min before; 2nd PET 2 indicates 60 min after FDOPA injection). (Values are

§ §*means6S.D.; , P,0.05; *, significant differences between group 1 and group 2; , significant differences within the related group compared with control1).

Table 2Transfer coefficients, distribution volume, and PS product of FDOPA of different brain regions calculated from the measured tracer activities in arterialblood and brain of newborn piglets during normoxia (1st PET) and normoxia (Group 1; n54) and hypoxia /hypercapnia (Group 2; n511) (2nd PET),respectively

FDOPA 21 21 FDOPA 21 FDOPA 21K (mL g min ) k (min ) k (min )1 2 3

Group 1 Group 2 Group 1 Group 2 Group 1 Group 2

Brain regionMesencephalon 1st PET 0.06260.011 0.07260.021 0.05760.020 0.09660.056 0.04760.038 0.06760.049

§2nd PET 0.05760.019 0.09360.058 0.05760.032 0.12760.090 0.03360.019 0.09560.064

Frontal cortex 1st PET 0.07860.032 0.06360.017 0.09660.029 0.06260.029 0.04360.009 0.05760.050§2nd PET 0.07260.030 0.08260.045 0.08360.029 0.09860.051 0.05260.013 0.07960.060

Striatum 1st PET 0.08060.035 0.07660.020 0.08160.012 0.09560.052 0.06960.018 0.06960.044§ §2nd PET 0.07060.029 0.09360.046 0.06360.018 0.12060.062 0.07260.017 0.09060.053

FDA1acids 21 FDOPA 21 FDOPA 21 21k (min ) V (mL g ) PS (mL g min )cl e

Mesencephalon 1st PET 0.01260.007 0.01160.014 1.2460.56 1.0360.60 0.06560.013 0.07760.0232nd PET 0.01860.005 0.01460.008 1.2960.12 0.8560.34 0.05960.020 0.09560.061

Frontal cortex 1st PET 0.00860.003 0.00860.013 0.8060.14 1.0960.60 0.08660.041 0.06760.0192nd PET 0.01060.002 0.01260.004 0.8760.14 0.9360.34 0.07760.034 0.08560.048

Striatum 1st PET 0.02860.019 0.01260.012 0.7960.13 1.1060.50 0.08760.042 0.07960.0192nd PET 0.02560.014 0.01360.007 0.9260.08 0.8960.36 0.07360.032 0.08460.026

§ § FDOPAValues are means6S.D.; P,0.05; indicates significant differences between 1st and 2nd PET measurement. K : unidirectional blood to brain1FDOPA FDOPA FDA1acidsclearance of FDOPA, k : rate constant for FDOPA backflux from the brain, k : apparent AADC activity, k : clearance rate constant for2 3 cl

FDOPA FDOPAFDOPA metabolites, V : distribution volume of FDOPA, PS : permeability–surface area product of FDOPA.e


respiratory and metabolic acidosis with a progressivelyincreased plasma lactate content (P,0.05). In terms ofbrain intracellular pH regulation, both components, how-ever, have to be considered differently. It has been shownthat in newborn piglets a gradual increase of arterial pCO2

and a concomitant gradual decrease of the arterial pH isimmediately followed by a proportional intracellular pHdecrease [13]. However, pure metabolic acidosis, causedby extracerebral delivery of organic acids such as lacticacid is obviously unable to decrease the intracellular brainpH [1]. Therefore, we assume that early after onset ofhypoxia /hypercapnia a significant decrease in intracellularpH occurred in the piglet brain. However, a further declinein brain intracellular pH remains unlikely, because CMRO2

was maintained and there was an increase of brain lactateuptake which is indicative of lactate metabolism, ratherthan production, in the brain.

Furthermore, whether an altered acid–base balance inthe brain may have a direct influence on cerebral FDOPAmetabolism in the newborn piglet must be considered.Firstly, there is no evidence that the combined respiratoryand metabolic acidosis induced by moderate hypoxia /hypercapnia may influence blood–brain transport of

18[ F]FDOPA. This is suggested because in this study nochange in K was found. Direct measurement of un-1

changed neutral amino acid uptake at neutral and acidicconditions in adult rats supports this interpretation [40].Secondly, the dependence of tyrosine hydroxylase (TH)Fig. 3. Relative amounts of FDOPA, fluorodopamine (FDA), 6-fluoro-activity from pH appears to be controlled by different3,4-dihydroxyphenylacetic acid (FDOPAC), 6-fluoro-3-methoxytyramine

(3-FMT), and 6-fluorohomovanillic acid (FHVA) in brain tissues protein phosphorylation sites. When an increase of TH(mesencephalon, frontal cortex and striatum) obtained from newborn activity was induced by increased cyclic AMP-dependentpiglets after normoxic control conditions (n54, filled columns) and phosphorylation, an alkaline shift in pH optimum (frompiglets exposed 120 min of moderate hypoxia /hypercapnia (n56, open

5.8–6.2 to 7.0–7.2) occurred [48]. In contrast, an increasecolumns). (Values are means6S.D.; *, P,0.05 indicates significantin TH activity by electrical stimulation of the medialdifferences between both groups).

forebrain bundle in rats did not change the optimal pH of5.8, but decreased the rate of decline in TH activity as thepH was increased above the optimum up to pH 7.5 [48]. Itappears difficult to speculate whether or not hypercapniclites (FMT, FDOPAC, FHVA) of all brain regions studiedhypoxia caused an alteration of TH phosphorylation. Awere significantly increased under H/H. The increasedfunctional activation may induce TH phosphorylation by aaccumulation of the acidic metabolites is indicative for ancalcium-activated kinase activity [21]. In contrast, in-increase in FDOPA metabolism, because of its time coursecreased glutamate release may decrease TH phosphoryla-in the newborn piglet brain [51]. In previous studies [3,9]tion via activation of NMDA receptors by reducing cyclicwe have described qualitatively similar PET findings usingAMP production [36]. Thirdly, in vitro studies have showndata obtained from a reduced animal number and analyzedthat only a small pH sensitivity of AADC between pH 5.5with a simplified kinetic model. This model did not includeand 7.5 exists [25,31]. Therefore we suggest that therea precise correction for the presence of the plasma-derived

metabolite 3-O-methyl-FDOPA based on separate mea- was—if any—apparently only a minimal impact of pHsurements of the kinetics of this compound. Obviously the change during moderate hypercapnic hypoxia on AADCuse of this simplified model provoked an underestimation activity.of the regional AADC activity during H/H. The additional An increase of dopaminergic AADC activity is notHPLC measurements of regional brain tissue contents of expected to be induced by brain O deficiency. We have2

fluorodopamine and the respective metabolites support the found that the CMRO remained unchanged throughout2

results of the PET study which indicates an elevation of the H/H period. Furthermore, MAP remained above thethe AADC activity during H/H. autoregulatory threshold throughout the period of moderate

Moderate hypercapnic hypoxia induced a combined hypoxia /hypercapnia [4,35] and an increased rate of brain


lactate metabolism occurred. This is not surprising, be- ‘trace’ amines act as fine tuning mechanisms allowingcause lactate appears to be an important metabolic sub- short-term regulation of monoaminergic tone without thestrate for the brain during the early period after birth and need for an alteration in the metabolic and degradativecan be used instead of other fuels such as glucose [28]. pathways of the transmitter itself [7]. Therefore, because of

Despite preserved cerebral O uptake, H/H obviously the increase in AADC activity, shown shortly after onset of2

provokes marked metabolic changes within the brain tissue moderate hypercapnic hypoxia, an increased dopaminergictogether with the markedly reduced brain tissue p , which activity should result consequently. This is proposed to beO2

seem to be involved in the increase of dopaminergic an independent effect in the alteration of dopaminergicAADC activity of newborn piglet brain. There is obviously activity owing to the moderate hypercapnic hypoxia inno oxygen reserve which protects the dopamine release addition to a possible increase of tyrosine hydroxylaseand metabolism from a decrease in oxygen pressure [29]. It which has been shown in newborn piglets after hypocapnicis assumed that a strong increase of [DA] should occur hypoxia [47].e

due to reduced brain tissue p of about one third of The hypothesis that an increase of [DA] during aO e2

hypoxic insult plays an important role in the pathogenesisnormoxic conditions. The H/H associated increase of theof neuronal injury in the newborn brain is supported bydopaminergic synthetic activity can also be caused by anumerous findings from different injury models. Directparallel activation of the glutamatergic system. An in-neurotoxic effects of DA on cultures have been showncreased glutamatergic activity has been suggested by an[43]. Furthermore, dopamine obviously plays an importantincreased oxygen consumption in the cerebral cortex androle in ischemia–reperfusion injury, because it has beenhypothalamus during moderate normocapnic hypoxia insuggested that an increase in extracellular dopamine cannewborn piglets which was blunted by glutamate receptorresult in alterations in the sensitivity of neurons to excitat-antagonists [54,55]. Furthermore, it has been shown thatory amino acids [23]. Another proposed mechanism for theglutamate receptor antagonists reduce hypoxia inducedneurotoxic effect of dopamine is through an increase in the[DA] increase in newborn piglets [34].e

production of free radicals. There are several pathways forOur findings support the assumption that the AADC isfree radical generation in the brain, to which dopaminean additional regulated step in the synthesis of DA [17].may contribute. Oxidation of the excess dopamine releasedAccumulated evidence suggests that AADC activity induring ischemia by molecular oxygen may occur duringadult brain is tuned by short- and long-term mechanismsreperfusion. Dopamine can react with hydroxyl radicals tothat apparently involve enzyme activation and induction.form the dopaminergic neurotoxin, 6-hydroxydopamine,Dopamine itself is able to modulate AADC activity. It haswhich generates free radicals during its spontaneous rapidbeen shown that the administration of dopamine D - and1

autooxidation [46]. Furthermore, the enzymatic oxidationD -like receptor blockers produced dose-dependent in-2

of dopamine by monoamine oxidase results also in thecreases in AADC activity [12,57] whereas mixed dopa-formation of hydrogen peroxide, a hydroxyl radical pre-mine receptor agonists inhibited AADC activity in ratcursor [37].striatal synaptosomes in a concentration- and time-depen-

Although a marked increase of the AADC activitydent manner [58]. Therefore, dopamine D - and D -re-1 2

occurred during H/H, the regional transport of FDOPA toceptors modulate AADC activity. A direct and transientFDOPA FDOPAthe brain indicated by K and PS was un-activation of AADC by dopamine loss is induced by a 1

changed. The PS product represents an indirect exponentialdirect phosphorylation of this enzyme by cyclic AMP [19],relation between actual blood perfusion rate and relatedwhich is obviously responsible for short-term modulationunidirectional rate constant of FDOPA blood–brain clear-of AADC activity. Subacute and chronic treatment with

FDOPAance (K ) [15]. If the relation between CBF anddopamine D - and D -receptor blockers caused induction 11 2FDOPAK is .8, as was found under baseline conditions,of AADC whereas only D -like agonists decreased AADC 12

then a further increase in CBF has only a marginal effect[12].FDOPAon PS decrease, e.g. under the precaution of un-Because AADC appears to be present in a large excess

FDOPAchanged K a CBF increase of more than 3-foldfor neurotransmitter synthesis in dopaminergic neurons, 1FDOPAthe effects of AADC regulation are suggested to be not induces an PS decrease ,5% of the baseline value.

predominantly relevant for the control of the overall flux in Therefore, under conditions of normal or increased brainthis pathway. It has been shown that AADC represents the perfusion, as shown in newborn piglets under isocapnic

FDOPAsole enzyme involved in ‘trace’ amine synthesis such as normoxia or moderate hypercapnic hypoxia, PS isFDOPA2-phenylethylamine and tryptamine synthesis [56], com- predominantly determined by K , which is unchanged1

pounds which have been suggested to act as endogenous under the conditions studied. Furthermore, the transportmodulators of monoamine neurotransmitters [42]. The process of FDOPA across the blood–brain barrier appears

FDOPAeffects of these compounds are seen as a limited, short- to be flow-independent, because at unchanged K 1

lasting, transient potentiation of the effect of the mono- regional blood flow increased during H/H (2nd PET scan)amine neurotransmitters. As a possible consequence, 3.4–4.1-fold compared to normoxic stage (1st PET scan).


¨[6] R. Bauer, B. Walter, E. Wurker, H. Kluge, U. Zwiener, ColouredIt is unlikely that the small but significant decrease inFDOPA microsphere technique as a new method for quantitative-multiplecortical and striatal V represents changes of thee estimation of regional hepatic and portal blood flow, Exp. Tox.

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