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Acute effects of perinatal hypoxic insult on concentrations of dopamine, serotonin, and metabolites in fetal monkey brain
@$Pergamon
hr. J. Dew/ Nerrroscimc~,
0736~5748(93)EOOOl-5
Vol. 12, No. 2. pp. 127-131. 1994 Elsevier Science Ltd. ISDN Printed in Great Britain 07_%-S74#94 $7.#+~.~)
ACUTE EFFECTS OF PERINATAL HYPOXIC INSULT ON CONCENTRATIONS OF DOPAMINE, SEROTONIN, AND METABOLITES IN FETAL MONKEY BRAIN ZBIGNIEW
BINIENDA,*
C. MAXTHEW
FOGLE, WILLIAM
SLIKKER,
JR and SYED F. ALI
Division of Neurotoxicology, National Center for Toxicological Research, Jefferson, AR 72079-9502, U.S.A. (Received 21 June 1993; revised 13 October 1993; accepted 18 October 1993)
Abstrad-Seven monkeys (Mncaca mulattu) were laparotomized under general anesthesia (halothane, nitrous oxide, oxygen). Fetal hypoxia was induced in four monkeys by occlusion of the umbilical cord with a hydraulic occluder for 5-6 min. Three sham-operated fetuses served as controls. After unclamping. the fetuses were allowed to reperfuse for 20-30 min. To monitor hypoxia. the fetal electrocardiogram was recorded continuously. Hypoxic insult was associated with a decrease in fetal heart rate during the occlusion. After reperfusion, fetuses were immediately sacrificed and neocortex regions dissected on ice, frozen on dry ice and stored at -70°C. Dopamine, 3,4-dihydroxyphenylacetic acid, homovanillic acid, serotonin, and 5-hydroxyindoleacetic acid were assayed by high performance liquid chromatography with electrochemical detection (HPLCJEC) in hip~campus, caudate nucleus and cortical regions. In the hippocampus, there was a significant increase in 5hydroxyindoleacetic acid concentration. In prefrontal cortex, there was a trend toward an increase in serotonin but no effects on dopamine and homovanillic acid concentrations. Dopamine, serotonin and metabolites were not altered in the caudate nucleus. These data demonstrate that fetal hypoxia followed by reperfusion produced an increase in serotonin concentration measured within the hippocampus and selected cortical areas known to be targets of hypoxic injury. Key words: monkey, hypoxia, fetus, brain, dopamine, serotonin.
death or various forms of long-term neurological motor deficits and learning disabilities.’ 1J5,17The mechanism of brain injury as a result of hypoxia is poorly understood. At least in part, hypoxic cerebral injury is mediated by excess release of the excitatory amino acids glutamate and aspartate.5xi4 The concentration of monoamines [i.e. dopamine (DA) and serotonin (5-HT)] in different brain regions also increases in response to ischemia-hypoxia.9~‘3~16 In adult rabbits, ische~a-hypoxia is associated with an increase of extracellular hippo~mpal con~ntrations of DA and 5-HT.4 The metabolism of monoamines, which increases during reoxygenation, may potentially result in formation of free radicals which could have a direct neurotoxic effect. On the other hand, we have previously shown that prenatal depletion of stores of S-HT due to reserpine action can cause a significant postnatal decrease in monoamine concentrations and DA receptor binding, correlated with behavioral changes such as slowed auditory startle habituation and decreased locomotor activity.1*7 There are no reports available on the effect of hypoxia on monoamine systems in nonhuman primates. The present experiment was undertaken to examine the acute effects of perinatal ischemia-hypoxia on the concentrations of DA, 5-m and their metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5hydroxyindoleacetic acid (S-HIAA) in near-term rhesus monkey brain regions which are known to be targets of hypoxic injury. Perinatal handicaps
cerebra1 hypoxia in~ludingepilepsy,
may lead to fetal mental retardation,
E~ERIMENTAL
PROCEDURES
Animals and surgical procedures
Seven pregnant rhesus monkeys were obtained from the NCTR nonhuman primate colony. The animals were individually-housed in a controlled environment (25&2”C, relative humidity 50t4%, *Author to whom correspondence should be addressed at: NCTR/FDA, Division of Neurotoxicology, Primate Research Facility. HFT-132.3900 NCTR Drive, Jefferson, AR 72079-9502, U.S.A. Abbreviations: DA, dopamine; S-HT, serotonin; DOPAC, 3,4-dihydroxyphenylacetic acid: HVA, homovanillic acid: 5-HIAA, S-hydroxyindoleacetic acid. 127
Z. Binienda et ~1.
12x
12 hr light/dark cycle; fed Purina Monkey Chow@ biscuits with ad lib water). Prior to anesthesia. the animals were injected intramuscularly (im.) with 0.01 mg/kg anticholinergic glycopyrrolate ( Robinol-V, A.H. Robins Company, Richmond. VA). and anesthetized with 10 mg/kg i.m. ketamine hydrochloride (Ketaset, Fort Dodge Laboratories, Inc., Fort Dodge, IA) . Surgery was performed under general gaseous anesthesia (1% halothane, 25% nitrous oxide, balance oxygen at 5 l/min) at gestational days 148-161 (term=approx. 165 days) as previously described.6The surgical procedure included laparotomy and hysterotomy followed by exposure of the fetus. In order to monitor fetal electrocardiogram (ECG) and heart rate, two ECG electrodes were placed on the fetal chest and thigh. The fetal ECG was recorded on a Grass Polygraph during the procedure. Blood samples (0.2 ml) were drawn by venipuncture to monitor fetal pH, acid/base status and blood gas tensions during hypoxia. A hydraulicoccluder RMI VO-4 (Rhodes Medical Instruments, Inc.. Woodland Hills. CA 91364) was installed around the umbilical cord next to the abdominal wall. The umbilical cord was occluded for 5-6 min in four fetuses. Three other fetuses, in which the occluder was not installed, served as controls. Fetuses were allowed to reperfuse for 20-30 min after the release of occlusion. After reperfusion, fetuses were immediately sacrificed by exsanguination. Their brains were quickly removed, dissected on ice, frozen on dry ice, and stored at -70°C for neurochemical analyses. DA, DOPAC, HVA, S-HT and 5-HIAA were determined in the caudate nucleus, hippocampus. prefrontal, frontal and entorhinal cortex as previously described.2 Briefly, the fetal monkey brains were dissected on an ice-cold plate using the atlas of Szebenyi ( 1970).2’ The cortex was removed as three pieces. The basal ganglia were dissected into the caudate nucleus and putamen and the hippocampi were rolled out from the surrounding temporal gyrus and frozen. As each sample was removed, it was immediately frozen on dry ice and each dissection was completed within 5 min ot removal of the brain from the animal. Neurochemical
anal~yses
Concentrations of DA, S-HT, DOPAC, HVA and 5-HIAA were quantitated by a high performance liquid chromatography (HPLC) combined with electrochemical detection as described previously. Briefly, each dissected brain region was weighed and diluted with a measured volume (10% W/V) of 0.2 N perchloric acid containing 100 r&ml of the internal standard 3,4_dihydrobenzylamine (DHBA). Brain tissue was then disrupted by ultrasonication, centrifuged (1000 g; 7 min) and 150 ml of the supernatant was removed and filtered through 0.2 ~.LMNylon-66 microfilter (MF-1 microcentrifuge filter, Bioanalytic System, BAS, W. Lafayette, IN). Aliquots of 25 t.r,lrepresenting 2.5 mg of brain tissue were injected directly onto the HPLClEC system for separation of the neurotransmitters and metabolites. Statistical analysis
The effect of occlusion of the umbilical circulation on neurochemistry of fetal brain regions following reperfusion was evaluated by two-way analysis of variance followed by Student’s t-test with the Bonferroni correction for multiple comparisons. The level of significance was assessed at PCO.05. RESULTS Fetal biophysical and cardiovascular response to occlusion of the umbilical circulation
Figure 1 shows normalized values of fetal blood pH, acid/base status and blood gas tensions in response to hypoxic-ischemic insult. Complete occlusion of umbilical circulation was accompanied by an immediate fall in fetal heart rate and irregularities in myocardial conduction seen as early as 40-50 set from the beginning of occlusion. Recovery of heart rate and ECG was observed after the release of occlusion. Changes in ECG and heart rate of a representative fetus are shown in Fig. 2. Fetal neurochemical
response to hypoxia
Two-way analysis of variance with region and treatment as factors revealed a significant effect of treatment (hypoxia-ischemia) for 5-HIAA (F’t2=5.55, P=O.O36) and nearly significant for 5-HT concentration (F’th=3.83. P=O.M8) in all areas. In particular, hypoxic-ischemic insult followed by
~onoa~ne
PH
levels in fetal hypoxic brain
PO2
129
Base
PC02
deftctt
Fig. 1. Normalized values of fetal blood pH, acid/base status and blood gas tensions at the end of the hypoxic insult expressed as a percentage of the baseline values (i.e. 100%). C]=Control, YJh=hypoxia. Mean ?SEM. *P
Fig. 2. Changes in heart rate (bradycardia) and ECG (extrasystole) seen during 6 min cord occlusion in fetal rhesus monkey Rh 8966.
produced a significant increase of 5-HIAA concentration (166% 517, P= 0.002; Table 1). The concentrations of 5-HT and 5-HIAA prefrontal cortex and entorhinal cortex, while 5-HT concentrations tended cortex. However, none of these trends reached statistical significance (Table
reperksion
in the hippo~~pus tended to increase in to increase in frontal 1). Concentrations of
Tabh 1. Acute effects of perinatal hypoxic insult followed by reperfusion on concentrations of serotonin (5HT) and 5-hydrox~henylacetic acid (SHIAA) in fetal monkey brain regions. Control n=3; hypoxia n=4. D=not detected. Data are means 5S.D. *P
Brain region Hippocampus control hypoxia Prefrontal cortex control hypoxia Frontal cortex control hypoxia Entorhinal cortex control hypoxia
5HT ng/lOO wet weight of tissue
5-HIAA ng/lOO mg wet weight of tissue
5.7 (2.3) 6.5 (1.3)
2.9 (0.4) 5.2 (0.6)*
2.3 (1.4) 6.3 (2.1)
2.1 (1.4) 5.2 (2.5)
1.8 (0.4) 2.7 (1.0)
ND
2.8 (2.3) 6.9 (8.2)
2.x (1.1) 3.7 (3.2)
2. Binienda rfd.
130
Table 2. Acute effects of perinatal hypoxic insult followrd by repcrfusion on concentrations of dopamine (DA), 3-~-d~hydroxyphenyia~tic acid (DOPAC) and nomovani~iic acid (HVA) in fetal monkey brain regions. Control ,z=3; ?iypoxia rr==4. ND=not detected. Data are means *SD. DA ng/lMI
mg
wet weight of tissue __. __.____--I
Brain region Caudate nucleus control hypoxia Frontal cortex control hypoxia
76-7 (366) 6X1 (310)
f)OPA(’ ng/lW mg wet weight of tissue ...._- . ..2h.9 2’3.3
74.l) (6.7) 71.2 (13.2)
HVA ngil(X) mp wet weight of tissue ______ ._..___--
(6.0) ( lh.3)
XL)
275 (6s) 251 (75) 1-I 5 (1.6) 15.7 (8.7)
DA and its metabolites (DOPAC and HVA) were not altered after hypoxic insult followed by reperfusion either in the caudate nucleus or the frontal cortex (Table 2).
DISCUSSION Occlusion of the umbilical cord produced disruption of fetal and placental circulation leading to oxygen deficit and acute fetal hypoxic stress. It has been shown that such stress may lead to permanent fetal brain damages with neurological and behavioral consequences in postnatal life.“” In the current study, there was a rapid fetal h~oxicresponse in blood gas tension and cardiovascular changes (i.e. bradycardia and alterations of ECG) due to complete umbilical cord occlusion. Thus. the physiologi~l effects in the current study very closely resemble those of other investigations of fetal hypoxia in the fetal baboon and rhesus monkey.‘8,2” Oxygen deprivation in utero is considered a major cause of neonatal encephalopathy and mental handicap in later life. 17,24It is thought that excitatory amino acid neurotransmitters are major mediators of the hypoxia-related neurotoxicity.5 Studies have shown, however, that ischemichypoxic insult is associated with an increase in excitatory (glutamate, aspartate) as well as inhibitory (y-aminoisobutyric acid, taurine) neurotransmitters in selected brain regions (e.g. hippocampus. striatum or the cortical regions). 4”10~13 Similarly, brain ischemia in rats and rabbits increased extracellular concentrations of DA and 5-HT. 4.9 In those studies utilizing in vivo microdialysis techniques, reperfusion following ischemia was accompanied by a reduction of extracellular mono~ines to the baseline ievel and an increase in the concentrations of their metabolites. In the present experiment. S-FIT, but not DA levels were elevated con~mitantly with 5HIAA in the cortex and hippocampus following ischemia-hypoxia. However, the technique applied in the present study allows measurement of total biogenic amines rather than extracellular concentrations. Also, the current technique does not allow for multiple sampling during hypoxia and recovery periods as is the case for in vivo microdialysis. Recently, it has been suggested that release of DA during ischemia-hypoxia may potentiate glutamate neurotoxicity mediated by activation of adenylate cyclase and increase of CAMP. I9 However, other monoamines including 5-HT may contribute to the activation of adenylate cyclase; thus, playing a role in enhancement of ischemia-hypoxia neurotoxicity.12 In the present study, increased concentration of S-HIAA in the hippocampus may indicate increased MAO-catalyzed metabolism of serotonin released during reoxygenation following ischemia-hypoxia. Degradation of S-F-ITto 5-HIAA is associated with formation of hydroxyl radicals22 which may be additive to the free radicals caused by hypoxic brain injury. In conclusion, the results of our study indicate an activation of serotonergic system during reoxygenation following fetal hypoxic insult. These changes in 5-HT and its MAO-catalyzed metabolism may contribute to neuronal damage frequently observed in ischemia-hypoxia and suggest vulnerability of fetal rhesus to hypoxia at term pregnancy.
AcknowlrdKrm~nrs-The procedures.
authors
would
like to acknowledge
Mr M. Gillam
for his technical
assistance
during
surgical
Monoa~ne
levels in fetal hypoxic brain
131
REFERENCES I. Ali S. F., Buelke-Sam J. and Slikker Jr. W. (1986) Prenatal reserpine exposure in rats decreases caudate nucleus dopamine receptor binding in female offspring. TOX. Lerr. 31,195-201. 2. Ali S. F., Newport G. D., Scallet A. C.. Binienda 2.. Ferguson S. A.. Bailey J. R.. Paule M. G. and Slikker Jr, W. (1993) Oral administration of 3,4-methylenediox~ethamphetamin~ (MDMA) produces selective serotonergic neurotoxi~ty in the nonhuman primate. Neurozox. Teratol. l&91-96. 3. Ali S. F.. David S. and Newport G. D. (1993) Age related susceptibility to MPTP-induced neurotoxicity. Nez~mtoxicolog.v I4 in press. 4. Baker A. J.. Zornow M. H., Scheller M. S.. Yaksh T. L.. Skilling S. R.. Smullin D. H., Larson A. A. and Kuczenski R. (1991) Changes in extracellular concentrations of glutamate, aspartate, glycine. dopamine. serotonin. and dopamine metaholites after transient global ischemia in the rabbit. J. Newochem. 57, 1370-1379. 5. Barks J. D. E. and Silverstain F. S. (1992) Excitatory amino acids contribute to the pathogenesis of perinatal hypoxic-is~hemi~ brain injury. Brain Pathol. 2,235-243. 6. Binienda Z., Bailey J. R.. Duhart l-l., Slikker Jr. W. and Paule M. G. (1993) Transplacental pharmacokinetics and maternal/fetal plasma concentrations of cocaine in pregnant macaques near term. Drzcg Metab. Disp. 21,36&368. 7. Buekle-Sam J., Ali S. F., Kimmel G. L., Slikker Jr, W.. Newport G. D. and Harmon J. R. (1989) Postnatal function following prenatal reserpine exposure in rats: Neurobehavioral toxicity. Neurotox. Terarof. ll,515-522. 8. Clapp J. F., Peress N. S., Wesley M. and Mann L. I. (1988) Brain damage after intermittent partial cord occlusion in the chronically instrumented fetal lamb. Am. J. Obstet. Gwzecol. 159, W-509. 9. Damsma G., Boisvert D. P.. Mudrick L. A., Wenkstern D. and Fibiger H. C. (1990) Effects of transient forebrain ischemia and pargyline on extracellular concentrations of dopamine, serotonin, and their metabolites in the rat striatum as determined by in viva microdialysis. J. Nezzrochem. 54,801-808. 10. Drejer J.. Benveniste H., Diemer N. H. and Schousboe A. (1985) Cellular origin of ischemia-induced glutamate release from brain tissue in viva and in virro. J. Neurochem. 45, 145-15 1. Il. Ellenberg J. H. and Nelson K. B. (1988) Cluster of perinatal events identifying infants as high risk for death or disability. J. Peffiutr. 113, 546-552. 12. Fujikura H., Kato H.. Nakano S. and Kogure K. (1989) A serotonin 52 antagonist, naftidrofuryl, exhibited a protective effect on ischemic neuronal damage in the gerbil. Brzzin Res. 4% 387-390. 13. Hagberg H.. Lehmann A., Sandberg M., Nystriim B., Jacobson 1. and Hamberger A. (1985) Ischemia-induced shift of inhibitory and excitatory amino acids from intra- to extracellular compartments. J. Cereb. Blood How Metab. 5,413-419. 14. Hill A. (1991) Current concepts of hypoxic-ischemic cerebral injury in the term newborn. Pediatr. Neural. 7,317-325. 15. Jensen F. E., Applegate C.. Burchfiel J. and Lombroso C. T. (1991) Differential effects of perinatal hypoxia and anoxia on long term seizure susceptibility in the rat. Lifr Sci. 49,399-407. 16. Kawano T., Tsutsumi K.. Mikyake H. and Mori K. (1988) Striatal dopamine in acute cerebral ischemia of stroke-resistant rats. Stroke 19, 1540-1543. 17. Low J. A., Galbraith R. S., Muir 13. W., Killen H. L.. Pater E. A. and Karchmar E. J. (1984) Factors associated with motor and cognitive deficits in children after intrapartum fetal hypoxia. Am. J. Obstet. Cynecol. 148, 533-539. 18. Mueller-Heubach E. and Battelli A. F. (1982) Variable heart rate decelerations and transcutaneous pOz during umbilical cord occlusion in fetal monkeys. Am. J. Ohs&t. G_ynecol. 144796802. 19. Prado R., Busto R. and Globus M. Y.-T. (1992) Ischemia-induced changes in extracellular levels of striatal cyclic AMP: Role of dopamine neurotransmission. J. Neurochem. 59,1581-1584. 20. Shen Y.. lsaacson R. L. and Smotherman W. P. (1991) The behavioral and anatomical effects of menatal umbilical cord clamping in the rat and their alteration by the prior maternal administration of nimodipine. R&. Neural. Nef~~oscj. 3, 1l-22. 21. Szebenyi E. S. (1970) Atlas ofMacaca mulatta. University Press, Rutherford, NJ. 22. Tipton K. F. (196X)The prosthetic groups of pig brain mitochondrial monoamine oxidase. Biochim. Bi0ph.y.r. Actu 159, 451459. 23. Yeh M.-N., Morishima H. O., Niemann W. H. and James L. S. (1975) Myocardial conduction defects in association with compression of the umbilical cord. Am. J. Obster. Gynecol. 121,951-957. 24. Younkin D. P. (1992) Hypoxic-ischemic brain injury of the newborn - statement of the problem and overview. Brzzin Pathof. 2.209-2 IO.
hr. J. Dew/ Nerrroscimc~,
0736~5748(93)EOOOl-5
Vol. 12, No. 2. pp. 127-131. 1994 Elsevier Science Ltd. ISDN Printed in Great Britain 07_%-S74#94 $7.#+~.~)
ACUTE EFFECTS OF PERINATAL HYPOXIC INSULT ON CONCENTRATIONS OF DOPAMINE, SEROTONIN, AND METABOLITES IN FETAL MONKEY BRAIN ZBIGNIEW
BINIENDA,*
C. MAXTHEW
FOGLE, WILLIAM
SLIKKER,
JR and SYED F. ALI
Division of Neurotoxicology, National Center for Toxicological Research, Jefferson, AR 72079-9502, U.S.A. (Received 21 June 1993; revised 13 October 1993; accepted 18 October 1993)
Abstrad-Seven monkeys (Mncaca mulattu) were laparotomized under general anesthesia (halothane, nitrous oxide, oxygen). Fetal hypoxia was induced in four monkeys by occlusion of the umbilical cord with a hydraulic occluder for 5-6 min. Three sham-operated fetuses served as controls. After unclamping. the fetuses were allowed to reperfuse for 20-30 min. To monitor hypoxia. the fetal electrocardiogram was recorded continuously. Hypoxic insult was associated with a decrease in fetal heart rate during the occlusion. After reperfusion, fetuses were immediately sacrificed and neocortex regions dissected on ice, frozen on dry ice and stored at -70°C. Dopamine, 3,4-dihydroxyphenylacetic acid, homovanillic acid, serotonin, and 5-hydroxyindoleacetic acid were assayed by high performance liquid chromatography with electrochemical detection (HPLCJEC) in hip~campus, caudate nucleus and cortical regions. In the hippocampus, there was a significant increase in 5hydroxyindoleacetic acid concentration. In prefrontal cortex, there was a trend toward an increase in serotonin but no effects on dopamine and homovanillic acid concentrations. Dopamine, serotonin and metabolites were not altered in the caudate nucleus. These data demonstrate that fetal hypoxia followed by reperfusion produced an increase in serotonin concentration measured within the hippocampus and selected cortical areas known to be targets of hypoxic injury. Key words: monkey, hypoxia, fetus, brain, dopamine, serotonin.
death or various forms of long-term neurological motor deficits and learning disabilities.’ 1J5,17The mechanism of brain injury as a result of hypoxia is poorly understood. At least in part, hypoxic cerebral injury is mediated by excess release of the excitatory amino acids glutamate and aspartate.5xi4 The concentration of monoamines [i.e. dopamine (DA) and serotonin (5-HT)] in different brain regions also increases in response to ischemia-hypoxia.9~‘3~16 In adult rabbits, ische~a-hypoxia is associated with an increase of extracellular hippo~mpal con~ntrations of DA and 5-HT.4 The metabolism of monoamines, which increases during reoxygenation, may potentially result in formation of free radicals which could have a direct neurotoxic effect. On the other hand, we have previously shown that prenatal depletion of stores of S-HT due to reserpine action can cause a significant postnatal decrease in monoamine concentrations and DA receptor binding, correlated with behavioral changes such as slowed auditory startle habituation and decreased locomotor activity.1*7 There are no reports available on the effect of hypoxia on monoamine systems in nonhuman primates. The present experiment was undertaken to examine the acute effects of perinatal ischemia-hypoxia on the concentrations of DA, 5-m and their metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5hydroxyindoleacetic acid (S-HIAA) in near-term rhesus monkey brain regions which are known to be targets of hypoxic injury. Perinatal handicaps
cerebra1 hypoxia in~ludingepilepsy,
may lead to fetal mental retardation,
E~ERIMENTAL
PROCEDURES
Animals and surgical procedures
Seven pregnant rhesus monkeys were obtained from the NCTR nonhuman primate colony. The animals were individually-housed in a controlled environment (25&2”C, relative humidity 50t4%, *Author to whom correspondence should be addressed at: NCTR/FDA, Division of Neurotoxicology, Primate Research Facility. HFT-132.3900 NCTR Drive, Jefferson, AR 72079-9502, U.S.A. Abbreviations: DA, dopamine; S-HT, serotonin; DOPAC, 3,4-dihydroxyphenylacetic acid: HVA, homovanillic acid: 5-HIAA, S-hydroxyindoleacetic acid. 127
Z. Binienda et ~1.
12x
12 hr light/dark cycle; fed Purina Monkey Chow@ biscuits with ad lib water). Prior to anesthesia. the animals were injected intramuscularly (im.) with 0.01 mg/kg anticholinergic glycopyrrolate ( Robinol-V, A.H. Robins Company, Richmond. VA). and anesthetized with 10 mg/kg i.m. ketamine hydrochloride (Ketaset, Fort Dodge Laboratories, Inc., Fort Dodge, IA) . Surgery was performed under general gaseous anesthesia (1% halothane, 25% nitrous oxide, balance oxygen at 5 l/min) at gestational days 148-161 (term=approx. 165 days) as previously described.6The surgical procedure included laparotomy and hysterotomy followed by exposure of the fetus. In order to monitor fetal electrocardiogram (ECG) and heart rate, two ECG electrodes were placed on the fetal chest and thigh. The fetal ECG was recorded on a Grass Polygraph during the procedure. Blood samples (0.2 ml) were drawn by venipuncture to monitor fetal pH, acid/base status and blood gas tensions during hypoxia. A hydraulicoccluder RMI VO-4 (Rhodes Medical Instruments, Inc.. Woodland Hills. CA 91364) was installed around the umbilical cord next to the abdominal wall. The umbilical cord was occluded for 5-6 min in four fetuses. Three other fetuses, in which the occluder was not installed, served as controls. Fetuses were allowed to reperfuse for 20-30 min after the release of occlusion. After reperfusion, fetuses were immediately sacrificed by exsanguination. Their brains were quickly removed, dissected on ice, frozen on dry ice, and stored at -70°C for neurochemical analyses. DA, DOPAC, HVA, S-HT and 5-HIAA were determined in the caudate nucleus, hippocampus. prefrontal, frontal and entorhinal cortex as previously described.2 Briefly, the fetal monkey brains were dissected on an ice-cold plate using the atlas of Szebenyi ( 1970).2’ The cortex was removed as three pieces. The basal ganglia were dissected into the caudate nucleus and putamen and the hippocampi were rolled out from the surrounding temporal gyrus and frozen. As each sample was removed, it was immediately frozen on dry ice and each dissection was completed within 5 min ot removal of the brain from the animal. Neurochemical
anal~yses
Concentrations of DA, S-HT, DOPAC, HVA and 5-HIAA were quantitated by a high performance liquid chromatography (HPLC) combined with electrochemical detection as described previously. Briefly, each dissected brain region was weighed and diluted with a measured volume (10% W/V) of 0.2 N perchloric acid containing 100 r&ml of the internal standard 3,4_dihydrobenzylamine (DHBA). Brain tissue was then disrupted by ultrasonication, centrifuged (1000 g; 7 min) and 150 ml of the supernatant was removed and filtered through 0.2 ~.LMNylon-66 microfilter (MF-1 microcentrifuge filter, Bioanalytic System, BAS, W. Lafayette, IN). Aliquots of 25 t.r,lrepresenting 2.5 mg of brain tissue were injected directly onto the HPLClEC system for separation of the neurotransmitters and metabolites. Statistical analysis
The effect of occlusion of the umbilical circulation on neurochemistry of fetal brain regions following reperfusion was evaluated by two-way analysis of variance followed by Student’s t-test with the Bonferroni correction for multiple comparisons. The level of significance was assessed at PCO.05. RESULTS Fetal biophysical and cardiovascular response to occlusion of the umbilical circulation
Figure 1 shows normalized values of fetal blood pH, acid/base status and blood gas tensions in response to hypoxic-ischemic insult. Complete occlusion of umbilical circulation was accompanied by an immediate fall in fetal heart rate and irregularities in myocardial conduction seen as early as 40-50 set from the beginning of occlusion. Recovery of heart rate and ECG was observed after the release of occlusion. Changes in ECG and heart rate of a representative fetus are shown in Fig. 2. Fetal neurochemical
response to hypoxia
Two-way analysis of variance with region and treatment as factors revealed a significant effect of treatment (hypoxia-ischemia) for 5-HIAA (F’t2=5.55, P=O.O36) and nearly significant for 5-HT concentration (F’th=3.83. P=O.M8) in all areas. In particular, hypoxic-ischemic insult followed by
~onoa~ne
PH
levels in fetal hypoxic brain
PO2
129
Base
PC02
deftctt
Fig. 1. Normalized values of fetal blood pH, acid/base status and blood gas tensions at the end of the hypoxic insult expressed as a percentage of the baseline values (i.e. 100%). C]=Control, YJh=hypoxia. Mean ?SEM. *P
Fig. 2. Changes in heart rate (bradycardia) and ECG (extrasystole) seen during 6 min cord occlusion in fetal rhesus monkey Rh 8966.
produced a significant increase of 5-HIAA concentration (166% 517, P= 0.002; Table 1). The concentrations of 5-HT and 5-HIAA prefrontal cortex and entorhinal cortex, while 5-HT concentrations tended cortex. However, none of these trends reached statistical significance (Table
reperksion
in the hippo~~pus tended to increase in to increase in frontal 1). Concentrations of
Tabh 1. Acute effects of perinatal hypoxic insult followed by reperfusion on concentrations of serotonin (5HT) and 5-hydrox~henylacetic acid (SHIAA) in fetal monkey brain regions. Control n=3; hypoxia n=4. D=not detected. Data are means 5S.D. *P
Brain region Hippocampus control hypoxia Prefrontal cortex control hypoxia Frontal cortex control hypoxia Entorhinal cortex control hypoxia
5HT ng/lOO wet weight of tissue
5-HIAA ng/lOO mg wet weight of tissue
5.7 (2.3) 6.5 (1.3)
2.9 (0.4) 5.2 (0.6)*
2.3 (1.4) 6.3 (2.1)
2.1 (1.4) 5.2 (2.5)
1.8 (0.4) 2.7 (1.0)
ND
2.8 (2.3) 6.9 (8.2)
2.x (1.1) 3.7 (3.2)
2. Binienda rfd.
130
Table 2. Acute effects of perinatal hypoxic insult followrd by repcrfusion on concentrations of dopamine (DA), 3-~-d~hydroxyphenyia~tic acid (DOPAC) and nomovani~iic acid (HVA) in fetal monkey brain regions. Control ,z=3; ?iypoxia rr==4. ND=not detected. Data are means *SD. DA ng/lMI
mg
wet weight of tissue __. __.____--I
Brain region Caudate nucleus control hypoxia Frontal cortex control hypoxia
76-7 (366) 6X1 (310)
f)OPA(’ ng/lW mg wet weight of tissue ...._- . ..2h.9 2’3.3
74.l) (6.7) 71.2 (13.2)
HVA ngil(X) mp wet weight of tissue ______ ._..___--
(6.0) ( lh.3)
XL)
275 (6s) 251 (75) 1-I 5 (1.6) 15.7 (8.7)
DA and its metabolites (DOPAC and HVA) were not altered after hypoxic insult followed by reperfusion either in the caudate nucleus or the frontal cortex (Table 2).
DISCUSSION Occlusion of the umbilical cord produced disruption of fetal and placental circulation leading to oxygen deficit and acute fetal hypoxic stress. It has been shown that such stress may lead to permanent fetal brain damages with neurological and behavioral consequences in postnatal life.“” In the current study, there was a rapid fetal h~oxicresponse in blood gas tension and cardiovascular changes (i.e. bradycardia and alterations of ECG) due to complete umbilical cord occlusion. Thus. the physiologi~l effects in the current study very closely resemble those of other investigations of fetal hypoxia in the fetal baboon and rhesus monkey.‘8,2” Oxygen deprivation in utero is considered a major cause of neonatal encephalopathy and mental handicap in later life. 17,24It is thought that excitatory amino acid neurotransmitters are major mediators of the hypoxia-related neurotoxicity.5 Studies have shown, however, that ischemichypoxic insult is associated with an increase in excitatory (glutamate, aspartate) as well as inhibitory (y-aminoisobutyric acid, taurine) neurotransmitters in selected brain regions (e.g. hippocampus. striatum or the cortical regions). 4”10~13 Similarly, brain ischemia in rats and rabbits increased extracellular concentrations of DA and 5-HT. 4.9 In those studies utilizing in vivo microdialysis techniques, reperfusion following ischemia was accompanied by a reduction of extracellular mono~ines to the baseline ievel and an increase in the concentrations of their metabolites. In the present experiment. S-FIT, but not DA levels were elevated con~mitantly with 5HIAA in the cortex and hippocampus following ischemia-hypoxia. However, the technique applied in the present study allows measurement of total biogenic amines rather than extracellular concentrations. Also, the current technique does not allow for multiple sampling during hypoxia and recovery periods as is the case for in vivo microdialysis. Recently, it has been suggested that release of DA during ischemia-hypoxia may potentiate glutamate neurotoxicity mediated by activation of adenylate cyclase and increase of CAMP. I9 However, other monoamines including 5-HT may contribute to the activation of adenylate cyclase; thus, playing a role in enhancement of ischemia-hypoxia neurotoxicity.12 In the present study, increased concentration of S-HIAA in the hippocampus may indicate increased MAO-catalyzed metabolism of serotonin released during reoxygenation following ischemia-hypoxia. Degradation of S-F-ITto 5-HIAA is associated with formation of hydroxyl radicals22 which may be additive to the free radicals caused by hypoxic brain injury. In conclusion, the results of our study indicate an activation of serotonergic system during reoxygenation following fetal hypoxic insult. These changes in 5-HT and its MAO-catalyzed metabolism may contribute to neuronal damage frequently observed in ischemia-hypoxia and suggest vulnerability of fetal rhesus to hypoxia at term pregnancy.
AcknowlrdKrm~nrs-The procedures.
authors
would
like to acknowledge
Mr M. Gillam
for his technical
assistance
during
surgical
Monoa~ne
levels in fetal hypoxic brain
131
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