- Email: [email protected]
Effects of asphyxia on the fetal lamb brain
Effects of asphyxia on the fetal lamb brain Harmen H. de Haan, MD: Jos L.H. Van Reempts," Johan S.H. VIes, MD, PhD,b Jelte de Haan, MD, PhD: and Tom H.M. Hasaart, MD, PhD" Maastricht, The Netherlands, and Beerse, Belgium OBJECTIVE: Our purpose was to study the effect of fetal asphyxia on the release of hypoxanthine and xanthine in cerebrospinal fluid and on brain histologic characteristics. STUDY DESIGN: In seven fetal lambs (3 to 5 days after surgery, gestational age 124.3 ± 2.6 days) asphyxia was induced by restriction of uterine blood flow. RESULTS: Fetal pH and base excess were reduced to 6.99 ± 0.02 and -17.6 ± 0.9 mmol/L, respectively. Cerebral blood flow increased during asphyxia and returned to normal in the recovery phase. Maximum concentrations of cerebrospinal fluid hypoxanthine and xanthine were reached in the normoxemic recovery phase. This high level of substrates during normoxemia facilitates oxygen free radical formation and may thus aggravate postasphyctic brain damage. Histologic evaluation of the brain 3 days after the insult showed a variable degree of edema. Coagulative neuronal changes, characteristic of irreversible cell death, were only occasionally detected. These changes were most obvious in the Purkinje cells of the cerebellum. CONCLUSIONS: Fetal asphyxia induced by uterine blood flow restriction is associated with high levels of cerebrospinal fluid hypoxanthine and xanthine in the recovery phase. Microscopically detectable brain damage, although not extensive, is mainly located in the cerebellum. (AM J OBSTET GYNECOL 1993;169:1493-501.)
Key words: Asphyxia, fetal lamb, brain histology, hypoxanthine, xanthine, cerebrospinal fluid
Fetal distress as a result of progressive fetal asphyxia can, if not corrected or circumvented, result in decompensation of physiologic responses and lead to permanent tissue injury or death. 1 At the level of the central nervous system this damage becomes clinically apparent in seizures, hypotonia, and motor and cognitive deficits.v" Several experimental setups have been used to investigate brain damage after oxygen shortage. Myers" 5 induced brain injury in neonatal monkeys. The animals were anesthetized or heavily sedated, and after asphyxia resuscitation was performed in a neonatal intensive care unit. The majority of monkeys, sustaining episodes of asphyxia severe enough to lead to widespread cerebral damage, also had injury to the myocardium, which resulted in death from cardiogenic shock within the first day or two after the insult.' Levine" circumvented that problem in adult rats by combining
From the Departments of Obstetrics and Gynecologya and Child Neurology/ University Hospital, and the Department of Life Sciences, Laboratory of Neuropathology, Janssen Research Foundation.' Presented in part at the Thirty-ninth Annual Meeting of the Society for Gynecologic Investigation, San Antonio, Texas, March 18-21, 1992, and at the Fortieth Annual Meeting of the Society for Gynecologic Investigation, Toronto, Ontario, Canada, March 31April 3, 1993. Reprints not available. Copyright © 1993 by Mosby-Year Book, 1nc. 0002-9378/93 $1.00 + .20 6/6/51428
unilateral carotid artery ligation with hypoxic exposure, thereby increasing the susceptibility for oxygen shortage in the ischemic cerebral hemisphere. In fetal lambs it seemed more difficult to obtain an adequate survival rate concomitant with an acceptable degree of histologic damage." The narrow threshold between a degree of intrauterine asphyxia associated with no sequelae, an insult causing persistent cerebral impairment, and an insult resulting in intrauterine death was confirmed. Recently, the "Levine model" was modified for fetal sheep. Ligation of the vertebral-carotid anastomosis and inflation of occluder cuffs around both carotid arteries for 30 minutes resulted in ischemic encephalopathy." However, although most setups described here led to reproducible and quantifiable brain damage, experimental cerebral ischemia is entirely different from the primarily hypoxic mechanism encountered in clinical practice. Therefore the pathophysiologic condition of acute fetal asphyxia was mimicked in this study by reducing maternal placental perfusion. The objective of the study was to induce severe asphyxia and evaluate its effect on (I) the fetal cardiovascular system and cerebral blood flow, (2) systemic and brain metabolic parameters (levels of cerebrospinal fluid lactate, hypoxanthine, and xanthine; the latter two are considered substrates in the formation of oxygen free radicals, involved in the induction of postasphyctic tissue damage"), and (3) histologic study of the brain after 3 days' survivaL 1493
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Material and methods
Surgery. Surgery was performed on seven pregnant Dutch Texel sheep of kno wn mating dates between 118 and 124 days' gestation (mean ± SO = 120.9 ± 2.0 days, term 147 days). General anes thes ia was induced with intravenous pentobarbital and maintained with 1% halothan e in a 2: 1 mixture of nitrou s oxide and oxygen . Before surgery the ewes received 1 gm of intravenou s ampicillin. Under sterile conditions a paramedian abdo minal incision was made, and an inflatable balloon occluder was placed around the maternal common internal iliac artery. Fetal in strumentation involved inser tion of polyvinyl catheters in th e axillary and femoral ar tery, the tips advanced to th e level of the brachioceph alic trunk and descending aorta, respectively. In addition, catheters were placed in th e femoral vein, the tip advan ced into the inferior ven a cava, and in th e amnio tic cavity. A polyvinyl cathe ter was placed in the fourth cerebral vent ricle, to obtai n cere bro spinal fluid. Cat he ters were exteriori zed to the ewe's flank . Ewes were housed in individu al cages with free access to food and water, and th ey were allowed to re cover from surgery for at least 3 da ys before the experiments were starte d. A continuou s slow infusion (I ml/hr) of heparin in saline solu tion (10 Uzrnl) was used to maintain paten cy of fetal arteri al and venous catheters. Guide lines for care and use of anima ls, as approved by the local Animal Medical Eth ics Co mmittee, were followed . Measurements. Fetal arterial blood pressure, fetal ven ou s blood pressure, and amniotic pressure were determined with the zero point at th e level of the ewe's spine . These signals, together with th e fetal heart rate derived from the pulsatile signa l of the femoral artery, were led to a bioelectric amplifier (Hewlett Packard 8800 series, Andover, Mass. ), di splayed on a monitor, recorded on eight-channel strip chart recorder, stored on magneti c tape, and digitized and ana lyzed with a compu ter. Regional cerebral blood flow was measured with radioactive microspheres with a diameter of 15 J.Lm. At random one of four available micro spheres (cerium 141 , ruthenium 103, niobium 95, and tin 113) was injec ted . Aggregation of micro spher es was prevented by ad ding Tween 80 to the inj ecti on medium. After homogenization in an ultrasonic waterba th at 39 C (Bransonic 5200, Soest, the Nethe rla nds) for 20 minutes, ap p roximately 0.5 x 106 microspheres were stirred on a vort ex agitator and infu sed into the inferior vena cava over 1 minute . Referen ce sampling (1.80 ml/rnin) (Harvard Apparatus, Kent, England) was started from the br achiocephalic ar ch 30 seconds before infusion, continued during, and stop ped 1 minute after infusion. Blood gas values and pH from th e fetal aortic arch 0
were me asured with an automated analyzer (AVL, Radi ometer, Copenhagen) and corr ected for 39 C. Hemoglobin saturation was me asured with a hernoximeter (OSM2 hemoximeter, Radiometer) . Arterial oxygen content was calculated .as follows: Arterial oxyge n content (rnmol/L) = Hemoglobin concen tration (rnmol/L) x Hemoglobin oxygen saturation (%/1 00). Immediately after a microsphere injection 2 ml of blood was withdrawn from the axillary artery and centrifuged (3 minutes at 13,000 revolutions/min). Serum was frozen in liquid nitrogen and stored at - 73 C. Samples of cerebrospinal fluid were withdrawn with a 1 ml syringe from the catheter in the fourth ventricle, centrifuged (3 minutes at 13,000 revolutions/min), and frozen in liquid nitrogen . The catheter deadspace was shortened to approximately 0.2 ml, and the volume of cerebros pinal fluid obtained per sample was 2: 0.6 m!. After stora ge at -73 C seru m and cerebrospinal fluid samples from all animals were analyzed together. Concen tra tions of lactate were mea sured, whereas levels of hypoxanthine and xanthine were determined by highp ressure liquid chromatography. " Experiments. Mean gestati onal age ( ± SO) during experiments was 124.3 (± 2.6) days. Fetal heart rate and arterial and veno us blood pressure were monitored continuo usly. Fetal acid-bas e state was analyzed every 15 minutes. To improve myocardial performance after asp hyxia, I I the fetal lambs received an intravenou s bolus of the adeno sine transport inhibiting drug R-75231 (2-[aminocarb onyl]-N-[4-amino-2,6-dichlorop henyl]-4-(5,5-bis[4-fluorophenyl]-pentyl)-I-piperazineacetamine, Janssen, Beersc, Belgium) in a dose of 0.1 mg/kg estimated fetal weight in the inferior vena cava before the onset of occlusion. Estimation of fetal weight was performed du ring instrumentation. After microsphere inje ction in th e baseline period fetal asp hyxia was induced . T o maintain the fetu s in a stable hem od ynamic cond ition, uterine blood flow was gr adually reduced by a ste pwise inflation of the balloon occlud er arou nd the common internal iliac artery over a period of approxim atel y 120 minutes, until fetal art erial oxygen content reached a value of 40 % of ba seline, in combination with an aortal pH < 7.15 . This moment was considered tim e = 0 minutes, and occlusion was kept at the same level for I hour. At time = 60 minutes, the nadir of asphyxia, microspheres were injected to calculate cerebral blo od flow. Thereafter the uterine blood flow ob struction was discontinued by emptying the balloon occluder. The third and fourth do sages of microspheres were administered in the recovery phase, at time = 90 minutes and time = 180 minutes, corresponding with 0.5 and 2 hours after release of the occluder, respectively. Fixation procedure and histologic evaluation. Three days after the period of asp hyxia a relaparotomy 0
0
0
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was performed with the animal under general anesthesia. The fetus was completely heparinized by 15,000 U intravenous heparin. The fetal heart was approached by median thoracotomy. A blunt steel catheter was inserted in the left ventricle. The right atrium was opened, and 500 ml of isotonic and buffered isocolloidal fluid (39 C) was infused. The descending aorta was clamped. The fetal brain was preserved by intracardial perfusion with Karnovsky's fixative (2% formaldehyde and 2.5% glutaraldehyde in phosphate buffer 0.1 mol/L, pH 7.40), approximately 1 L at room temperature. The fetus was weighed, and correct catheter placement was confirmed. After l-day immersion fixation in Karnovsky's fixative the fetal brain was removed from the skull and was sampled for cerebral blood flow determination and histologic analysis, according to a stereotaxic atlas of the ovine fetal brain." After scintillation counting, vibratome sections (200 urn) were cut from eight cerebral areas (frontal cortex, parietal cortex, temporal cortex, striatum, hippocampus, cerebellum, thalamus, and medulla oblongata). Sections were postfixed in 2% osmium tetroxide, buffered with barbital acetate (0.05 mol/L, pH 7.40), dehydrated in graded series of ethanol, and routinely embedded in Epon. Light microscopic evaluation was performed on 2 urn sections stained with toluidine blue. Fixation quality was scored on a 10-point scale" whereby 10 corresponds with preservation in which no residual erythrocytes are detected, with open microvessels and without disseminated cell swelling. A 0 corresponds with a morphologic picture dominated by collapsed microvessels filled with residual erythrocytes and surrounded by spongy brain parenchyma. Two characteristic types of hypoxic cell changes were evaluated: edematous cell changes, mainly involving the glial compartment, and coagulative neuronal cell changes. Cell changes were also scored on a lO-point scale, whereby o is absence of coagulative changes and lOis 100% of the tissue damaged. 13 Calculations and data analysis. Arterial blood pressure, corrected for amniotic pressure, was averaged over 10-second periods with a computer program. Five of these epochs were averaged to calculate mean arterial blood pressure. Samples from the eight cerebral areas were weighed (± 1 gm), put into test tubes, and preserved in 3% glutaraldehyde solution. Radioactivity in all samples was determined before histologic evaluation. All four isotopes were counted simultaneously. Radioactivity in tissue and in reference samples was measured with an automatic ')I-scintillation counter and sample changer system (analyzer model 45, Molsgaard, Horsholm, Denmark), connected to a ND680 programable analyzercomputer system (Nuclear Data, Frankfurt, Germany). Parameters during asphyxia and in the recovery pe0
riod were compared with baseline values by means of a Friedman analysis of variance, with time as the repeated measure. A p value < 0.05 was taken to represent statistical significance. Results
The time course of mean arterial oxygen content, pH, and arterial blood pressure is depicted in Fig. 1. Arterial oxygen content decreased gradually, and was approximately 35% of baseline level during asphyxia. During the occlusion period the mean pH decreased from 7.36 to 6.99. Release of the occluder resulted in recovery, with a mean pH of 7.22 at 180 minutes. Arterial blood pressure remained unaltered during the entire experiment. Table I summarizes the fetal arterial oxygen content, pH, Pcoj, base excess, serum glucose levels, and serum lactate concentrations. Glucose levels remained unaltered during the experiment, but serum lactate levels rose during asphyxia more than eightfold compared with baseline levels (Friedman two-way analysis of variance, p < 0.05), and remained elevated during the hours monitored in the recovery phase. All fetal lambs in this study were successfully instrumented with a catheter in the fourth cerebral ventricle. It was not always possible to maintain patency of these catheters, and therefore cerebrospinal fluid could be collected in five preparations only. Because of sampling problems cerebrospinal fluid could not be obtained on standardized moments. The results of cerebrospinal fluid analysis are depicted in Fig. 2. During the hour of severe asphyxia concentrations of lactate, hypoxanthine, and xanthine started to rise, which continued during the first hours of recovery. Maximum values of hypoxanthine and xanthine in cerebrospinal fluid were found approximately at 180 minutes. Thereafter the concentrations started to return to normal. Cerebrospinal fluid lactate levels, however, remained more or less unchanged during the 3 hours after asphyxia, at a level of approximately 9 mmol/L. The weight-specific regional cerebral blood flow (in milliliters per minute x 100 gm) is shown in Table II. During asphyxia cerebral blood flow in all anatomic areas increased, whereas normalization occurred in the recovery period. All seven fetuses recovered and were in a good metabolic condition the day after asphyxia. One ewe had contractions and the fetus died during labor. The period between intrauterine fetal death and birth resulted in a poor quality of fixation, which made reliable histologic evaluation impossible (Table III, lamb No.5). Another fetal lamb bled to death because of a catheter accident. The brain of this animal was immersion fixed; it showed typical morphologic signs of inadequate fixation (e.g., a high amount of residual erythrocytes,
1496 de Haan et al.
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(mmol/L)
4.0 _ arterial oxygen content
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time in min Fig. 1. Time course of arterial oxygen content (in mmol/L), pH and arterial blood pressure (in mm Hg), n = 7, expressed as mean ± SEM. Time -180 to -120 min, Baseline period; time -120 to 0 min, progressive reduction of uterine blood flow; time 0 to 60 min, 1 hour period of asphyxia; time 60 to 180 min, recovery period; bar, period of occlusion.
extreme swelling of the perivascular and perineuronal glial compartment, densification of neuronal cell bodies, and granular appearance of nuclear chromatin) (Table III, lamb No.4). It was impossible to differentiate these changes from subtle experimental hypoxic damage. Five fetuses survived for 72 hours after asphyxia. This 3-day period was chosen to allow delayed
neuronal cell death to become histologically manifest. 14, 15 The metabolic condition of these five animals during the whole experiment did not differ from that of the two animals lost for perfusion fixation because of labor and hemorrhage, The brains of the five fetuses were adequately perfused (Fig, 3, Table III), Structural abnormalities in the brains of these survivors were
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Fig. 2. Cerebrospinal fluid (CSF) levels (n = 5) of lactate in millimoles per liter (upper panel),
hypoxanthine in micromoles per liter (middlepanel), and xanthine in micromoles per liter (lowerpanel) during experiment. All five animals are represented by specific symbol. Time -180 to -120 min, Baseline period; time -120 to 0 min, progressive reduction of uterine blood flow; time 0 to 60 min, I hour period of asphyxia; time 60 to 240 min, recovery period; bar, period of occlusion.
Table I. Fetal acid-base state and serum glucose and lactate levels Baseline Arterial oxygen content
3.31 ± 0.42
Asphyxia 1.56 ± 0.16*
Time
=
90 min
2.80 ± 0.41
Time = 180 min 2.91 ± 0.45
(rnmol/L)
pH Pco 2 (kPa) Base excess (mmol/L) Serum glucose (mmol/L) Serum lactate (mmol/L)
7.36 5.15 -4.1 1.05 1.8
± ± ± ± ±
0.01 0.20 0.8 0.14 0.5
6.99 7.37 -17.6 0.96 14.7
± ± ± ± ±
0.02* 0.33* 0.9* 0.18 0.6*
7.08 6.14 -15.6 0.96 14.4
± ± ± ± ±
0.01 * 0.19* 0.3* 0.09 0.6*
7.22 ± 5.57 ± -9.4± 1.02 ± 11.5 ±
0.03* 0.19 1.7* 0.10 0.7*
Values are expressed as mean ± SEM, n = 7. Asphyxia is at time = 60 min, at the nadir of asphyxia. Time = 90 min and time = 180 min correspond with 0.5 and 2 hours after release of the occluder, respectively. *P < 0.05 (asphyxia, time = 90 min, or time = 180 min vs baseline, Friedman two-way analysis of variance).
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Fig. 3. Section of adequately fixed fetal cortex 72 hours after period of severe asphyxia. Microvessels appear nicely expanded (arrows) and are surrounded by compact neuropil. Neurons (N) and glial cells (arrowhead) are well preserved, as can be derived from absence of any coagulative or edematous cell changes (2 urn, toluidine blue).
Fig. 4. Detail of fetal cerebellum 72 hours after period of severe asphyxia. Purkinje cells (P) show characteristic coagulative cell change with cytoplasmic microvacuolation (arrow) and nuclear pyknosis (arrowhead). Surrounding glial cells are extremely dilated (asterisk). Granular cell layer (GL) and molecular cell layer (ML) appear unaltered (2 urn, toluidine blue).
Table II. Regional cerebral blood flow Baseline
Frontal cortex Parietal cortex Temporal cortex Striatum Hippocampus Thalamus Cerebellum Medulla oblongata
135 129 133 156 151 234 208 292
± ± ± ± ± ± ± ±
20 22 30 23 27 52 34 69
Asphyxia
278 288 295 362 372 546 476 776
± ± ± ± ± ± ± ±
Time
55* 59* 57* 57* 79* 80* 63* 118*
= 90
143 ± 134 ± 122 ± 156 ± 154 ± 239 ± 252 ± 326 ±
min
26 22 23 28 34 50 36 70
Time
= 180 min
154 ± 157 ± 141 ± 202 ± 173 ± 291 ± 281 ± 357 ±
34 42 36 45 57 85 63 94
Values are expressed in millimeters per minute x 100 gm and as mean ± SEM, n = 7. Asphyxia is at time = 60 min, at the nadir of asphyxia. Time = 90 min and time = 180 min correspond with 0.5 and 2 hours after release of the occluder, respectively. *P < 0.05 (asphyxia vs baseline, Friedman two-way analysis of variance).
scarce. No changes were present that could be attributed to microsphere embolism. A variable degree of edema was found in white and gray matter. Coagulative neuronal cell changes, characteristic of irreversible cell death,": 14 were only occasionally detected in four animals (Table III). These changes were most obvious in the Purkinje cells of the cerebellum (Fig. 4).
Comment Reduction of uterine perfusion with an inflatable balloon occluder resulted in decreased fetal arterial oxygen content, the onset of anaerobic metabolism, the production of lactate, and a decrease in pH. Mean arterial blood pressure was 4?0 ± 0.6 mm Hg and
remained at the same level during and after asphyxia (Fig. 1). This is in agreement with previous data.": 17 Arterial blood pressure gathers importance because intermittent increases in cerebral intravascular pressure during asphyxia caused hemorrhages in the germinal layer of fetal sheep brain." On the other hand, hypotension also has been shown to be associated with neuronal damage." 19 During asphyxia adenosine is released primarily in the myocardium. Adenosine is considered as a natural cardioprotector because of its coronary vasodilative and antiadrenergic effects and its inhibition of the activation of platelets and Ieukocytes.!" Pretreatment with the adenosine transport inhibiting drug R-75231 therefore
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Table III. Degree of fixation and histologic evaluation
Lamb No.
Survival (hr)
Degree of fixation
1 2 3 4 5 6 7
72 72 72 24 22
6.0 5.3 7.7 0.2 NP
72
9.0 7.6
72
Edematous cell changes str 4 3 1 4 NP 1
0
I
etx 6 2 1 4 NP 1 1
I
thZ 6 1 0 4 NP
0 0
I
hip 2 4 1 6 NP NP
0
Coagulative cell changes
I
eer
I
I
med
str
etx
4 4 0 6 NP
1 0 0 4 NP
0 0 0 ? NP
0 3 0 ? NP
0 1 0 0 NP
0 0
0 NP
0 0
0 0
0 0
IthZlhiPI
eer
I
med
0 0 0 0 NP NP
0 4 2 4 NP 1
0 0 0 1 NP
0
0
NP
0
Histologic cell changes were scored in eight cerebral entities: str, striatum; thl, thalamus; hip, hippocampus; cer, cerebellum; and med, medulla oblongata, whereas etx summarizes the results of evaluation of the frontal, parietal, and temporal cortex. Ni; Not performed. Details for the scoring of the degree of fixation and histologic brain damage.":
preserves the function of the myocardium when the heart has been compromised by severe asphyxia II and was shown to improve myocardial functioning after heart transplantations in dogs." Therefore we hypothesized that after pretreatment with R-75231 the degree of asphyxia might be severe enough to cause cerebral damage without the concomitant fetal death from cardiogenic injury. Baseline values of cerebral blood flow were in agreement with previous results" and data." Cerebral blood flow increased in the period of asphyxia. During asphyxia the increase in cerebral blood flow was greater in phylogenetic older structures, such as medulla oblongata and cerebellum, than in the cortex. In the current study fetuses remained normoglycemic (Table I) when compared with literature data on glucose levels." 22, 23 Apparently the fetuses were able to maintain normal glucose concentrations in spite of severe asphyxia. Hyperglycemia, shown to reduce the tolerance of the brain toward asphyxia," was not detected in any fetal lamb. The potentially negative impact on the brain of (experimentally induced) high levels of glucose during oxygen lack was ascribed to the concomitant tissue acidosis." Therefore in the current study brain damage is not explained by an additional hyperglycemia. After release of the occluder the arterial oxygen content returned to normal, leading to recovery of the fetal acid-base state. The time course of recovery after this degree of asphyxia is in agreement with data from other investigators." Cerebrospinal fluid lactate levels still increased during this recovery phase (Fig. 2). Maximum concentrations were reached approximately 2 hours after the end of uterine blood flow obstruction. After this moment there appeared to be a trend toward normalization. Cerebrospinal fluid concentrations of hypoxanthine and xanthine (Fig. 2), both catabolites of adenosine 5' -triphosphate, started to increase during the hour of severe asphyxia. At time = 60 minutes, the start of the recovery period, levels of cerebrospinal fluid
14
are provided in the text.
lactate, hypoxanthine, and xanthine were only slightly elevated. However, during the recovery period the concentration of these catabolites continued to rise. Maximum concentrations (fivefold, fivefold, and fourfold the baseline levels of cerebrospinal fluid lactate, hypoxanthine, and xanthine, respectively) were reached at approximately time = 180 minutes. Next to damage to the blood-brain-eerebrospinal fluid compartment barrier, delayed neuronal damage may also partly explain the increase of hypoxanthine and xanthine in the recovery period." During this phase the arterial oxygen content returned to baseline levels. The enzyme xanthine oxidase is released under hypoxic conditions from the liver into the circulation" and is present in the brain." This enzyme metabolizes hypoxanthine and xanthine to excretable uric acid, during which process oxygen free radicals are formed. 26, 27 High levels of hypoxanthine and xanthine in the brain, in combination with normal levels of oxygen in the recovery phase, may facilitate oxygen free radical formation and aggravate postasphyctic brain damage." This could explain the beneficial effect of oxygen free radical scavengers in various experiments with asphyxiated animals.": 29 Although in the current study the degree of asphyxia was severe, as indicated by a mean pH < 7.00, serum lactate levels > 14 mmol/L, and a base excess > - 17 mmol/L (Table I, Fig. 1), extensive histologic brain damage was not observed. Cerebral edema was present in the majority of fetuses. From Table III it can be derived that the degree of fixation was inversely correlated with edematous cell changes. However; the question remains if this edema is a postmortem effect after incomplete fixation or if the incomplete fixation is a result of edema induced by the asphyctic insult. The latter is supported by combining the cerebrospinal fluid hypoxanthine and xanthine data with the results of histologic studies. Animal No.1 (Table III) is depicted in Fig. 2 with open circles; edematous cell changes at histologic evaluation corresponded with the highest
1500 de Haan et al.
level of cerebrospinal fluid lactate and a long period of elevated levels of cerebrospinal fluid hypoxanthine. Animal No. 2 is depicted with filled circles in Fig. 2. Edematous cell changes are present throughout the whole cerebrum in the fetus that had the highest amount of cerebrospinal fluid hypoxanthine and xanthine (Fig. 2). Cerebellar Purkinje cells were shown to be most sensitive to oxygen deprivation (Fig. 4). These cells have highly branched dendritic trees that receive a huge amount of synaptic input from axon terminals. During asphyxia these large cells may encounter a nutrient supply smaller than their minimum needs, resulting in irreversible cell death. The sensitivity of Purkinje cells for oxygen shortage was confirmed in experiments in other species. In adult cat brain chronic hypoxia mostly affected the Purkinje cells located in the deeper portions of cerebellar folia." After severe asphyxia and resuscitation most newborn pigs showed some anoxic cerebellar damage, with scattered necrotic Purkinje cells." In the latter two experiments and in the current study the insult was primarily hypoxic, and damage was predominantly located in the cerebellum. This is in contrast to brain ischemic experiments, which primarily result in damage of the parasagittal cortex." This cortical area is considered to be a watershed area: an arterial end field in the border zone between the territories of major cerebral arteries. During ischemia the relative sparing of midbrain and hindbrain structures such as the cerebellum, when compared with the cortex, may reflect preferential perfusion of the residual normoxemic blood flow to these structures." In sheep various methods were used to reduce fetal oxygen delivery to induce brain damage; umbilical cord occlusion led to hippocampal brain damage" and common internal iliac artery occlusion led to parasagittal cortex and striatum darnage.!'' In the latter study damage was not extensive: only three of the 14 surviving fetuses demonstrated > 10% damage and six of the 14 animals showed no damage at all. The authors presumed that differences in cerebral lactate concentration could be an explanation for the regional differences in neuronal loss between the two studies, but neither neuronal nor cerebrospinal fluid lactate levels were measured. The lack of extensive brain damage reported after various types of cerebral hypoxia confirms the statement about the existence of a narrow margin between lethality after asphyxia, on the one hand, and survival without signs of histologic damage, on the other hand;" Finally, adequate perfusion fixation is an absolute necessity, especially because the brain is extremely sensitive to postmortem changes and because these changes closely resemble lesions induced by mild hypoxia." In summary, restriction of maternal uterine blood flow resulted in increased fetal serum lactate concentra-
December 1993 Am J Obstet Gynecol
tions and severe asphyxia. During asphyxia animals increased their cerebral blood flow and remained normotensive and normoglycemic. Cerebrospinal fluid concentrations of lactate, hypoxanthine, and xanthine increased fivefold, fivefold, and fourfold, respectively. Maximum concentrations of hypoxanthine and xanthine were reached in the recovery period after asphyxia and were associated with edematous cell changes. In the recovery period the arterial oxygen content returned to baseline values. This reoxygenation may facilitate oxygen free radical formation and may thus aggravate postasphyctic brain damage. In distinguishing between histologic damage as a result of asphyxia or as a postmortem artifact, perfusion fixation appeared to be important. The absence of extensive histologic brain damage indicates that, under normotensive and normoglycemic conditions, the fetal brain can tolerate severe asphyxia. Cerebellar Purkinje cells tended to be most sensitive to asphyxia. We thank May Bost, Joyce Suyk, Jan Geilen, and Marc Haseldonckx for skillful technical assistance; Anke IJ zermans for assistance in performing the experiments; Frans Slangen and Peter Franssen for animal care; and Guy .Jacobs for preparation of the photographs. REFERENCES 1. Parer JT, Livingston EG. What is fetal distress? AM J OBSTET GVNECOL 1990;162:1421-7. 2. Low JA, Galbraith RS, Muir DW, Killen HL, Pater EA, Karchmar EJ. Motor and cognitive deficits after intrapartum asphyxia in the mature fetus. AM J OBSTET GVNECOL 1988; 158:356-61. 3. Goodwin TM, Belai I, Hernandez P, Durand M, Paul RH. Asphyxial complications in the term newborn with severe umbilical acidemia. AMJ OBSTET GYNECOL 1992;167:150612. 4. Myers RE. Two patterns of perinatal brain damage and their conditions of occurrence. AM J OBSTET GVNECOL 1972; 112:246-76. 5. Myers RE. Four patterns of perinatal brain damage and their conditions of occurrence in primates. Adv Neural 1975; 10:223-34. 6. Levine S. Anoxic-ischemic encephalopathy in rats. Am J PathoI1960;36:1-17. 7. Ting P, Yamaguchi S, Bacher JD, Killens RH, Myers RE. Hypoxic-ischemic cerebral necrosis in midgestational sheep fetuses: physiopathologic correlations. Exp Neural 1983;80:227-45. 8. Williams CE, Gunn AJ, Synek B, Gluckman PD. Delayed seizures occurring with hypoxic-ischemic encephalopathy in the fetal sheep. Pediatr Res 1990;27:561-5. 9. Saugstad OD. Hypoxanthine as an indicator of hypoxia: its role in health and disease through free radical production. Pediatr Res 1988;23: 143-50. 10. Van Belle H, Goossens F, Wynants J. Formation and release of purine catabolites during hypoperfusion, anoxia, and ischemia. Am J Physiol 1987;252:H886-93. 11. de Haan HH, de Haan J, Van Reempts JLH, Van Belle H, Hasaart THM. The effect of adenosine transport inhibition on cardiovascular function and survival after severe asphyxia in fetal lambs. Pediatr Res 1993;33:185-9. 12. Gluckman PD, Parsons Y. Stereotaxic neurosurgery on the ovine fetus. In: Nathanielsz PW, ed. Animal models in fetal
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13. 14.
15. 16.
17. 18.
19. 20.
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22.
23.
medicine, part 3. New York: Perinatology Press, 1984:69107. Van Reempts ]LH. The hypoxic brain: histological and ultrastructural aspects. Behav Brain Res 1984; 14:99-1 08. Van Reempts JLH, Borgers M. Structural damage in experimental cerebral ischemia. In: Schurr A, Rigor BM, eds. Cerebral ischemia and resuscitation. Boca Raton, Florida: CRC, 1990:235-57. Pulsinelli WA, Brierley JB, Plum JB. Temporal profile of neuronal damage in a model of transient forebrain ischemia. Ann Neurol 1982;11:491-8. de Haan HH, Van Reempts JLH, Borgers M, de Haan J, Vies ]SH, Hasaart THM. Possible neuroprotective properties of flunarizine infused after severe asphyxia in fetal lambs are not explained by effects on cerebral blood flow or systemic blood pressure. Pediatr Res 1993;34:37984. Field DR, Parer JT, Auslender RA, Cheek DB, Baker W, Johnson]. Cerebral oxygen consumption during asphyxia in fetal sheep.] Dev PhysioI1990;14:131-7. Reynolds ML, Evans CAN, Reynolds EOR, Saunders NR, Durbin GM, Wigglesworth JS. Intracranial haemorrhage in the preterm sheep fetus. Early Hum Dev 1979;3: 16386. Gunn A], Parer JT, Mallard EC, Williams CE, Gluckman PD. Cerebral histologic and electrocorticographic changes after asphyxia in fetal sheep. Pediatr Res 1992;31:486-91. Flameng W, Sukehiro S, M611hoffT, Van Belle H, Janssen PA]. A new concept of long-term donor heart preservation: nucleoside transport inhibition. J Heart Lung Transplant 1992; I 0:990-8. Peeters LLH, Sheldon RE, Jones MD, Makowski EL, Meschia G. Blood flow to fetal organs as a function of arterial oxygen content. AM J OBSTET GYNECOL 1979;135: 637-46. Blomstrand S, Hrbek A, Karlsson K, Kjellmer I, Lindecrantz K, Olsson T. Does glucose administration affect the cerebral response to fetal asphyxia? Acta Obstet Gynecol Scand 1984;63:345-53. Thiringer K, Blomstrand S, Hrbek A, Karlsson K, Kjellmer I. Cerebral arterio-venous difference for hypoxanthine and lactate during graded asphyxia in the fetal lamb. Brain Res 1982;239:107-17.
de Haan et al. 1501
24. de Courten-Myers GM, Kleinholz M, Wagner KR, Myers RE. Fatal stroke in hyperglycemic cats. Stroke 1989;20: 1707-15. 25. Kjellmer I, Andine P, Hagberg H, Thiringer K. Extracellular increase of hypoxanthine and xanthine in the cortex and basal ganglia of fetal lambs during hypoxia-ischemia. Brain Res 1989;478:241-7. 26. McCordJM. Oxygen-derived free radicals in postischemic tissue injury. N Engl] Med 1985;312:159-63. 27. Vannucci RC. Experimental biology of cerebral hypoxiaischemia: relation to perinatal brain damage. Pediatr Res 1990;27:317-26. 28. Thiringer K, Hrbek A, Karlsson K, Rosen KG, Kjellmer I. Postasphyxial cerebral survival in newborn sheep after treatment with oxygen free radical scavengers and a calcium antagonist. Pediatr Res 1987;22:62-6. 29. Rosenberg AA, Murdaugh E, White CWo The role of oxygen free radicals in postasphyxia cerebral hypoperfusion in newborn lambs. Pediatr Res 1989;26:215-9. 30. Cervos-NavarroJ, Sampaolo S, HamdorfG. Brain changes in experimental chronic hypoxia. Exp Pathol 1991;42: 205-12. 31. Rootwelt T, Loberg EM, Moen A, Oyasaeter S, Saugstad OD. Hypoxemia and reoxygenation with 21% or 100% oxygen in newborn pigs: changes in blood pressure, base deficit, and hypoxanthine and brain morphology. Pediatr Res 1992;32:107-13. 32. Ashwal S, Dale PS, Longo LD. Regional cerebral blood flow: studies in the fetal lamb during hypoxia, hypercapnia, acidosis, and hypotension. Pediatr Res 1984;18:130916. 33. Mallard EC, Gunn AJ, Williams CE, Johnston BM, Gluckman PD. Transient umbilical cord occlusion causes hippocampal damage in the fetal sheep. AM J OBSTET GYNECOL 1992; 167: 1423-30. 34. Hicks SP. Brain metabolism in vivo. I. The distribution of lesions caused by cyanide poisoning, insulin hypoglycemia, asphyxia in nitrogen and fluoroacetate poisoning in rats. Arch Pathol 1950;49: 111-37. 35. Cammermeyer]. The importance of avoiding "dark" neurons in experimental neuropathology. Acta Neuropathol 1961; I :245-70.
Key words: Asphyxia, fetal lamb, brain histology, hypoxanthine, xanthine, cerebrospinal fluid
Fetal distress as a result of progressive fetal asphyxia can, if not corrected or circumvented, result in decompensation of physiologic responses and lead to permanent tissue injury or death. 1 At the level of the central nervous system this damage becomes clinically apparent in seizures, hypotonia, and motor and cognitive deficits.v" Several experimental setups have been used to investigate brain damage after oxygen shortage. Myers" 5 induced brain injury in neonatal monkeys. The animals were anesthetized or heavily sedated, and after asphyxia resuscitation was performed in a neonatal intensive care unit. The majority of monkeys, sustaining episodes of asphyxia severe enough to lead to widespread cerebral damage, also had injury to the myocardium, which resulted in death from cardiogenic shock within the first day or two after the insult.' Levine" circumvented that problem in adult rats by combining
From the Departments of Obstetrics and Gynecologya and Child Neurology/ University Hospital, and the Department of Life Sciences, Laboratory of Neuropathology, Janssen Research Foundation.' Presented in part at the Thirty-ninth Annual Meeting of the Society for Gynecologic Investigation, San Antonio, Texas, March 18-21, 1992, and at the Fortieth Annual Meeting of the Society for Gynecologic Investigation, Toronto, Ontario, Canada, March 31April 3, 1993. Reprints not available. Copyright © 1993 by Mosby-Year Book, 1nc. 0002-9378/93 $1.00 + .20 6/6/51428
unilateral carotid artery ligation with hypoxic exposure, thereby increasing the susceptibility for oxygen shortage in the ischemic cerebral hemisphere. In fetal lambs it seemed more difficult to obtain an adequate survival rate concomitant with an acceptable degree of histologic damage." The narrow threshold between a degree of intrauterine asphyxia associated with no sequelae, an insult causing persistent cerebral impairment, and an insult resulting in intrauterine death was confirmed. Recently, the "Levine model" was modified for fetal sheep. Ligation of the vertebral-carotid anastomosis and inflation of occluder cuffs around both carotid arteries for 30 minutes resulted in ischemic encephalopathy." However, although most setups described here led to reproducible and quantifiable brain damage, experimental cerebral ischemia is entirely different from the primarily hypoxic mechanism encountered in clinical practice. Therefore the pathophysiologic condition of acute fetal asphyxia was mimicked in this study by reducing maternal placental perfusion. The objective of the study was to induce severe asphyxia and evaluate its effect on (I) the fetal cardiovascular system and cerebral blood flow, (2) systemic and brain metabolic parameters (levels of cerebrospinal fluid lactate, hypoxanthine, and xanthine; the latter two are considered substrates in the formation of oxygen free radicals, involved in the induction of postasphyctic tissue damage"), and (3) histologic study of the brain after 3 days' survivaL 1493
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Material and methods
Surgery. Surgery was performed on seven pregnant Dutch Texel sheep of kno wn mating dates between 118 and 124 days' gestation (mean ± SO = 120.9 ± 2.0 days, term 147 days). General anes thes ia was induced with intravenous pentobarbital and maintained with 1% halothan e in a 2: 1 mixture of nitrou s oxide and oxygen . Before surgery the ewes received 1 gm of intravenou s ampicillin. Under sterile conditions a paramedian abdo minal incision was made, and an inflatable balloon occluder was placed around the maternal common internal iliac artery. Fetal in strumentation involved inser tion of polyvinyl catheters in th e axillary and femoral ar tery, the tips advanced to th e level of the brachioceph alic trunk and descending aorta, respectively. In addition, catheters were placed in th e femoral vein, the tip advan ced into the inferior ven a cava, and in th e amnio tic cavity. A polyvinyl cathe ter was placed in the fourth cerebral vent ricle, to obtai n cere bro spinal fluid. Cat he ters were exteriori zed to the ewe's flank . Ewes were housed in individu al cages with free access to food and water, and th ey were allowed to re cover from surgery for at least 3 da ys before the experiments were starte d. A continuou s slow infusion (I ml/hr) of heparin in saline solu tion (10 Uzrnl) was used to maintain paten cy of fetal arteri al and venous catheters. Guide lines for care and use of anima ls, as approved by the local Animal Medical Eth ics Co mmittee, were followed . Measurements. Fetal arterial blood pressure, fetal ven ou s blood pressure, and amniotic pressure were determined with the zero point at th e level of the ewe's spine . These signals, together with th e fetal heart rate derived from the pulsatile signa l of the femoral artery, were led to a bioelectric amplifier (Hewlett Packard 8800 series, Andover, Mass. ), di splayed on a monitor, recorded on eight-channel strip chart recorder, stored on magneti c tape, and digitized and ana lyzed with a compu ter. Regional cerebral blood flow was measured with radioactive microspheres with a diameter of 15 J.Lm. At random one of four available micro spheres (cerium 141 , ruthenium 103, niobium 95, and tin 113) was injec ted . Aggregation of micro spher es was prevented by ad ding Tween 80 to the inj ecti on medium. After homogenization in an ultrasonic waterba th at 39 C (Bransonic 5200, Soest, the Nethe rla nds) for 20 minutes, ap p roximately 0.5 x 106 microspheres were stirred on a vort ex agitator and infu sed into the inferior vena cava over 1 minute . Referen ce sampling (1.80 ml/rnin) (Harvard Apparatus, Kent, England) was started from the br achiocephalic ar ch 30 seconds before infusion, continued during, and stop ped 1 minute after infusion. Blood gas values and pH from th e fetal aortic arch 0
were me asured with an automated analyzer (AVL, Radi ometer, Copenhagen) and corr ected for 39 C. Hemoglobin saturation was me asured with a hernoximeter (OSM2 hemoximeter, Radiometer) . Arterial oxygen content was calculated .as follows: Arterial oxyge n content (rnmol/L) = Hemoglobin concen tration (rnmol/L) x Hemoglobin oxygen saturation (%/1 00). Immediately after a microsphere injection 2 ml of blood was withdrawn from the axillary artery and centrifuged (3 minutes at 13,000 revolutions/min). Serum was frozen in liquid nitrogen and stored at - 73 C. Samples of cerebrospinal fluid were withdrawn with a 1 ml syringe from the catheter in the fourth ventricle, centrifuged (3 minutes at 13,000 revolutions/min), and frozen in liquid nitrogen . The catheter deadspace was shortened to approximately 0.2 ml, and the volume of cerebros pinal fluid obtained per sample was 2: 0.6 m!. After stora ge at -73 C seru m and cerebrospinal fluid samples from all animals were analyzed together. Concen tra tions of lactate were mea sured, whereas levels of hypoxanthine and xanthine were determined by highp ressure liquid chromatography. " Experiments. Mean gestati onal age ( ± SO) during experiments was 124.3 (± 2.6) days. Fetal heart rate and arterial and veno us blood pressure were monitored continuo usly. Fetal acid-bas e state was analyzed every 15 minutes. To improve myocardial performance after asp hyxia, I I the fetal lambs received an intravenou s bolus of the adeno sine transport inhibiting drug R-75231 (2-[aminocarb onyl]-N-[4-amino-2,6-dichlorop henyl]-4-(5,5-bis[4-fluorophenyl]-pentyl)-I-piperazineacetamine, Janssen, Beersc, Belgium) in a dose of 0.1 mg/kg estimated fetal weight in the inferior vena cava before the onset of occlusion. Estimation of fetal weight was performed du ring instrumentation. After microsphere inje ction in th e baseline period fetal asp hyxia was induced . T o maintain the fetu s in a stable hem od ynamic cond ition, uterine blood flow was gr adually reduced by a ste pwise inflation of the balloon occlud er arou nd the common internal iliac artery over a period of approxim atel y 120 minutes, until fetal art erial oxygen content reached a value of 40 % of ba seline, in combination with an aortal pH < 7.15 . This moment was considered tim e = 0 minutes, and occlusion was kept at the same level for I hour. At time = 60 minutes, the nadir of asphyxia, microspheres were injected to calculate cerebral blo od flow. Thereafter the uterine blood flow ob struction was discontinued by emptying the balloon occluder. The third and fourth do sages of microspheres were administered in the recovery phase, at time = 90 minutes and time = 180 minutes, corresponding with 0.5 and 2 hours after release of the occluder, respectively. Fixation procedure and histologic evaluation. Three days after the period of asp hyxia a relaparotomy 0
0
0
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was performed with the animal under general anesthesia. The fetus was completely heparinized by 15,000 U intravenous heparin. The fetal heart was approached by median thoracotomy. A blunt steel catheter was inserted in the left ventricle. The right atrium was opened, and 500 ml of isotonic and buffered isocolloidal fluid (39 C) was infused. The descending aorta was clamped. The fetal brain was preserved by intracardial perfusion with Karnovsky's fixative (2% formaldehyde and 2.5% glutaraldehyde in phosphate buffer 0.1 mol/L, pH 7.40), approximately 1 L at room temperature. The fetus was weighed, and correct catheter placement was confirmed. After l-day immersion fixation in Karnovsky's fixative the fetal brain was removed from the skull and was sampled for cerebral blood flow determination and histologic analysis, according to a stereotaxic atlas of the ovine fetal brain." After scintillation counting, vibratome sections (200 urn) were cut from eight cerebral areas (frontal cortex, parietal cortex, temporal cortex, striatum, hippocampus, cerebellum, thalamus, and medulla oblongata). Sections were postfixed in 2% osmium tetroxide, buffered with barbital acetate (0.05 mol/L, pH 7.40), dehydrated in graded series of ethanol, and routinely embedded in Epon. Light microscopic evaluation was performed on 2 urn sections stained with toluidine blue. Fixation quality was scored on a 10-point scale" whereby 10 corresponds with preservation in which no residual erythrocytes are detected, with open microvessels and without disseminated cell swelling. A 0 corresponds with a morphologic picture dominated by collapsed microvessels filled with residual erythrocytes and surrounded by spongy brain parenchyma. Two characteristic types of hypoxic cell changes were evaluated: edematous cell changes, mainly involving the glial compartment, and coagulative neuronal cell changes. Cell changes were also scored on a lO-point scale, whereby o is absence of coagulative changes and lOis 100% of the tissue damaged. 13 Calculations and data analysis. Arterial blood pressure, corrected for amniotic pressure, was averaged over 10-second periods with a computer program. Five of these epochs were averaged to calculate mean arterial blood pressure. Samples from the eight cerebral areas were weighed (± 1 gm), put into test tubes, and preserved in 3% glutaraldehyde solution. Radioactivity in all samples was determined before histologic evaluation. All four isotopes were counted simultaneously. Radioactivity in tissue and in reference samples was measured with an automatic ')I-scintillation counter and sample changer system (analyzer model 45, Molsgaard, Horsholm, Denmark), connected to a ND680 programable analyzercomputer system (Nuclear Data, Frankfurt, Germany). Parameters during asphyxia and in the recovery pe0
riod were compared with baseline values by means of a Friedman analysis of variance, with time as the repeated measure. A p value < 0.05 was taken to represent statistical significance. Results
The time course of mean arterial oxygen content, pH, and arterial blood pressure is depicted in Fig. 1. Arterial oxygen content decreased gradually, and was approximately 35% of baseline level during asphyxia. During the occlusion period the mean pH decreased from 7.36 to 6.99. Release of the occluder resulted in recovery, with a mean pH of 7.22 at 180 minutes. Arterial blood pressure remained unaltered during the entire experiment. Table I summarizes the fetal arterial oxygen content, pH, Pcoj, base excess, serum glucose levels, and serum lactate concentrations. Glucose levels remained unaltered during the experiment, but serum lactate levels rose during asphyxia more than eightfold compared with baseline levels (Friedman two-way analysis of variance, p < 0.05), and remained elevated during the hours monitored in the recovery phase. All fetal lambs in this study were successfully instrumented with a catheter in the fourth cerebral ventricle. It was not always possible to maintain patency of these catheters, and therefore cerebrospinal fluid could be collected in five preparations only. Because of sampling problems cerebrospinal fluid could not be obtained on standardized moments. The results of cerebrospinal fluid analysis are depicted in Fig. 2. During the hour of severe asphyxia concentrations of lactate, hypoxanthine, and xanthine started to rise, which continued during the first hours of recovery. Maximum values of hypoxanthine and xanthine in cerebrospinal fluid were found approximately at 180 minutes. Thereafter the concentrations started to return to normal. Cerebrospinal fluid lactate levels, however, remained more or less unchanged during the 3 hours after asphyxia, at a level of approximately 9 mmol/L. The weight-specific regional cerebral blood flow (in milliliters per minute x 100 gm) is shown in Table II. During asphyxia cerebral blood flow in all anatomic areas increased, whereas normalization occurred in the recovery period. All seven fetuses recovered and were in a good metabolic condition the day after asphyxia. One ewe had contractions and the fetus died during labor. The period between intrauterine fetal death and birth resulted in a poor quality of fixation, which made reliable histologic evaluation impossible (Table III, lamb No.5). Another fetal lamb bled to death because of a catheter accident. The brain of this animal was immersion fixed; it showed typical morphologic signs of inadequate fixation (e.g., a high amount of residual erythrocytes,
1496 de Haan et al.
December 1993 Am J Obstet Gynecol
(mmol/L)
4.0 _ arterial oxygen content
T
T
•1
3.0
•1
- T T
~·.T ill. -
T T
••1 •1T •1
1
T
1
2.0
1.0
,
7.40
r
730t 7.20
I
pH
• •
~
... - -
-
.. -
I 1 7.00 I
7.10
• • T
-
-
T
!
.1.
•
1.
_
•
•
.
.L
•
•
6.90L,-~~~~~~~~--+-----;-
50
40
.'
T _
•-
T
J.
•1
30L-~~~~~~~~--+--120 -180 120 -60 60 o
I ,
-
-I
180
time in min Fig. 1. Time course of arterial oxygen content (in mmol/L), pH and arterial blood pressure (in mm Hg), n = 7, expressed as mean ± SEM. Time -180 to -120 min, Baseline period; time -120 to 0 min, progressive reduction of uterine blood flow; time 0 to 60 min, 1 hour period of asphyxia; time 60 to 180 min, recovery period; bar, period of occlusion.
extreme swelling of the perivascular and perineuronal glial compartment, densification of neuronal cell bodies, and granular appearance of nuclear chromatin) (Table III, lamb No.4). It was impossible to differentiate these changes from subtle experimental hypoxic damage. Five fetuses survived for 72 hours after asphyxia. This 3-day period was chosen to allow delayed
neuronal cell death to become histologically manifest. 14, 15 The metabolic condition of these five animals during the whole experiment did not differ from that of the two animals lost for perfusion fixation because of labor and hemorrhage, The brains of the five fetuses were adequately perfused (Fig, 3, Table III), Structural abnormalities in the brains of these survivors were
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Fig. 2. Cerebrospinal fluid (CSF) levels (n = 5) of lactate in millimoles per liter (upper panel),
hypoxanthine in micromoles per liter (middlepanel), and xanthine in micromoles per liter (lowerpanel) during experiment. All five animals are represented by specific symbol. Time -180 to -120 min, Baseline period; time -120 to 0 min, progressive reduction of uterine blood flow; time 0 to 60 min, I hour period of asphyxia; time 60 to 240 min, recovery period; bar, period of occlusion.
Table I. Fetal acid-base state and serum glucose and lactate levels Baseline Arterial oxygen content
3.31 ± 0.42
Asphyxia 1.56 ± 0.16*
Time
=
90 min
2.80 ± 0.41
Time = 180 min 2.91 ± 0.45
(rnmol/L)
pH Pco 2 (kPa) Base excess (mmol/L) Serum glucose (mmol/L) Serum lactate (mmol/L)
7.36 5.15 -4.1 1.05 1.8
± ± ± ± ±
0.01 0.20 0.8 0.14 0.5
6.99 7.37 -17.6 0.96 14.7
± ± ± ± ±
0.02* 0.33* 0.9* 0.18 0.6*
7.08 6.14 -15.6 0.96 14.4
± ± ± ± ±
0.01 * 0.19* 0.3* 0.09 0.6*
7.22 ± 5.57 ± -9.4± 1.02 ± 11.5 ±
0.03* 0.19 1.7* 0.10 0.7*
Values are expressed as mean ± SEM, n = 7. Asphyxia is at time = 60 min, at the nadir of asphyxia. Time = 90 min and time = 180 min correspond with 0.5 and 2 hours after release of the occluder, respectively. *P < 0.05 (asphyxia, time = 90 min, or time = 180 min vs baseline, Friedman two-way analysis of variance).
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December 1993 Am J Obstet Gynecol
Fig. 3. Section of adequately fixed fetal cortex 72 hours after period of severe asphyxia. Microvessels appear nicely expanded (arrows) and are surrounded by compact neuropil. Neurons (N) and glial cells (arrowhead) are well preserved, as can be derived from absence of any coagulative or edematous cell changes (2 urn, toluidine blue).
Fig. 4. Detail of fetal cerebellum 72 hours after period of severe asphyxia. Purkinje cells (P) show characteristic coagulative cell change with cytoplasmic microvacuolation (arrow) and nuclear pyknosis (arrowhead). Surrounding glial cells are extremely dilated (asterisk). Granular cell layer (GL) and molecular cell layer (ML) appear unaltered (2 urn, toluidine blue).
Table II. Regional cerebral blood flow Baseline
Frontal cortex Parietal cortex Temporal cortex Striatum Hippocampus Thalamus Cerebellum Medulla oblongata
135 129 133 156 151 234 208 292
± ± ± ± ± ± ± ±
20 22 30 23 27 52 34 69
Asphyxia
278 288 295 362 372 546 476 776
± ± ± ± ± ± ± ±
Time
55* 59* 57* 57* 79* 80* 63* 118*
= 90
143 ± 134 ± 122 ± 156 ± 154 ± 239 ± 252 ± 326 ±
min
26 22 23 28 34 50 36 70
Time
= 180 min
154 ± 157 ± 141 ± 202 ± 173 ± 291 ± 281 ± 357 ±
34 42 36 45 57 85 63 94
Values are expressed in millimeters per minute x 100 gm and as mean ± SEM, n = 7. Asphyxia is at time = 60 min, at the nadir of asphyxia. Time = 90 min and time = 180 min correspond with 0.5 and 2 hours after release of the occluder, respectively. *P < 0.05 (asphyxia vs baseline, Friedman two-way analysis of variance).
scarce. No changes were present that could be attributed to microsphere embolism. A variable degree of edema was found in white and gray matter. Coagulative neuronal cell changes, characteristic of irreversible cell death,": 14 were only occasionally detected in four animals (Table III). These changes were most obvious in the Purkinje cells of the cerebellum (Fig. 4).
Comment Reduction of uterine perfusion with an inflatable balloon occluder resulted in decreased fetal arterial oxygen content, the onset of anaerobic metabolism, the production of lactate, and a decrease in pH. Mean arterial blood pressure was 4?0 ± 0.6 mm Hg and
remained at the same level during and after asphyxia (Fig. 1). This is in agreement with previous data.": 17 Arterial blood pressure gathers importance because intermittent increases in cerebral intravascular pressure during asphyxia caused hemorrhages in the germinal layer of fetal sheep brain." On the other hand, hypotension also has been shown to be associated with neuronal damage." 19 During asphyxia adenosine is released primarily in the myocardium. Adenosine is considered as a natural cardioprotector because of its coronary vasodilative and antiadrenergic effects and its inhibition of the activation of platelets and Ieukocytes.!" Pretreatment with the adenosine transport inhibiting drug R-75231 therefore
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Table III. Degree of fixation and histologic evaluation
Lamb No.
Survival (hr)
Degree of fixation
1 2 3 4 5 6 7
72 72 72 24 22
6.0 5.3 7.7 0.2 NP
72
9.0 7.6
72
Edematous cell changes str 4 3 1 4 NP 1
0
I
etx 6 2 1 4 NP 1 1
I
thZ 6 1 0 4 NP
0 0
I
hip 2 4 1 6 NP NP
0
Coagulative cell changes
I
eer
I
I
med
str
etx
4 4 0 6 NP
1 0 0 4 NP
0 0 0 ? NP
0 3 0 ? NP
0 1 0 0 NP
0 0
0 NP
0 0
0 0
0 0
IthZlhiPI
eer
I
med
0 0 0 0 NP NP
0 4 2 4 NP 1
0 0 0 1 NP
0
0
NP
0
Histologic cell changes were scored in eight cerebral entities: str, striatum; thl, thalamus; hip, hippocampus; cer, cerebellum; and med, medulla oblongata, whereas etx summarizes the results of evaluation of the frontal, parietal, and temporal cortex. Ni; Not performed. Details for the scoring of the degree of fixation and histologic brain damage.":
preserves the function of the myocardium when the heart has been compromised by severe asphyxia II and was shown to improve myocardial functioning after heart transplantations in dogs." Therefore we hypothesized that after pretreatment with R-75231 the degree of asphyxia might be severe enough to cause cerebral damage without the concomitant fetal death from cardiogenic injury. Baseline values of cerebral blood flow were in agreement with previous results" and data." Cerebral blood flow increased in the period of asphyxia. During asphyxia the increase in cerebral blood flow was greater in phylogenetic older structures, such as medulla oblongata and cerebellum, than in the cortex. In the current study fetuses remained normoglycemic (Table I) when compared with literature data on glucose levels." 22, 23 Apparently the fetuses were able to maintain normal glucose concentrations in spite of severe asphyxia. Hyperglycemia, shown to reduce the tolerance of the brain toward asphyxia," was not detected in any fetal lamb. The potentially negative impact on the brain of (experimentally induced) high levels of glucose during oxygen lack was ascribed to the concomitant tissue acidosis." Therefore in the current study brain damage is not explained by an additional hyperglycemia. After release of the occluder the arterial oxygen content returned to normal, leading to recovery of the fetal acid-base state. The time course of recovery after this degree of asphyxia is in agreement with data from other investigators." Cerebrospinal fluid lactate levels still increased during this recovery phase (Fig. 2). Maximum concentrations were reached approximately 2 hours after the end of uterine blood flow obstruction. After this moment there appeared to be a trend toward normalization. Cerebrospinal fluid concentrations of hypoxanthine and xanthine (Fig. 2), both catabolites of adenosine 5' -triphosphate, started to increase during the hour of severe asphyxia. At time = 60 minutes, the start of the recovery period, levels of cerebrospinal fluid
14
are provided in the text.
lactate, hypoxanthine, and xanthine were only slightly elevated. However, during the recovery period the concentration of these catabolites continued to rise. Maximum concentrations (fivefold, fivefold, and fourfold the baseline levels of cerebrospinal fluid lactate, hypoxanthine, and xanthine, respectively) were reached at approximately time = 180 minutes. Next to damage to the blood-brain-eerebrospinal fluid compartment barrier, delayed neuronal damage may also partly explain the increase of hypoxanthine and xanthine in the recovery period." During this phase the arterial oxygen content returned to baseline levels. The enzyme xanthine oxidase is released under hypoxic conditions from the liver into the circulation" and is present in the brain." This enzyme metabolizes hypoxanthine and xanthine to excretable uric acid, during which process oxygen free radicals are formed. 26, 27 High levels of hypoxanthine and xanthine in the brain, in combination with normal levels of oxygen in the recovery phase, may facilitate oxygen free radical formation and aggravate postasphyctic brain damage." This could explain the beneficial effect of oxygen free radical scavengers in various experiments with asphyxiated animals.": 29 Although in the current study the degree of asphyxia was severe, as indicated by a mean pH < 7.00, serum lactate levels > 14 mmol/L, and a base excess > - 17 mmol/L (Table I, Fig. 1), extensive histologic brain damage was not observed. Cerebral edema was present in the majority of fetuses. From Table III it can be derived that the degree of fixation was inversely correlated with edematous cell changes. However; the question remains if this edema is a postmortem effect after incomplete fixation or if the incomplete fixation is a result of edema induced by the asphyctic insult. The latter is supported by combining the cerebrospinal fluid hypoxanthine and xanthine data with the results of histologic studies. Animal No.1 (Table III) is depicted in Fig. 2 with open circles; edematous cell changes at histologic evaluation corresponded with the highest
1500 de Haan et al.
level of cerebrospinal fluid lactate and a long period of elevated levels of cerebrospinal fluid hypoxanthine. Animal No. 2 is depicted with filled circles in Fig. 2. Edematous cell changes are present throughout the whole cerebrum in the fetus that had the highest amount of cerebrospinal fluid hypoxanthine and xanthine (Fig. 2). Cerebellar Purkinje cells were shown to be most sensitive to oxygen deprivation (Fig. 4). These cells have highly branched dendritic trees that receive a huge amount of synaptic input from axon terminals. During asphyxia these large cells may encounter a nutrient supply smaller than their minimum needs, resulting in irreversible cell death. The sensitivity of Purkinje cells for oxygen shortage was confirmed in experiments in other species. In adult cat brain chronic hypoxia mostly affected the Purkinje cells located in the deeper portions of cerebellar folia." After severe asphyxia and resuscitation most newborn pigs showed some anoxic cerebellar damage, with scattered necrotic Purkinje cells." In the latter two experiments and in the current study the insult was primarily hypoxic, and damage was predominantly located in the cerebellum. This is in contrast to brain ischemic experiments, which primarily result in damage of the parasagittal cortex." This cortical area is considered to be a watershed area: an arterial end field in the border zone between the territories of major cerebral arteries. During ischemia the relative sparing of midbrain and hindbrain structures such as the cerebellum, when compared with the cortex, may reflect preferential perfusion of the residual normoxemic blood flow to these structures." In sheep various methods were used to reduce fetal oxygen delivery to induce brain damage; umbilical cord occlusion led to hippocampal brain damage" and common internal iliac artery occlusion led to parasagittal cortex and striatum darnage.!'' In the latter study damage was not extensive: only three of the 14 surviving fetuses demonstrated > 10% damage and six of the 14 animals showed no damage at all. The authors presumed that differences in cerebral lactate concentration could be an explanation for the regional differences in neuronal loss between the two studies, but neither neuronal nor cerebrospinal fluid lactate levels were measured. The lack of extensive brain damage reported after various types of cerebral hypoxia confirms the statement about the existence of a narrow margin between lethality after asphyxia, on the one hand, and survival without signs of histologic damage, on the other hand;" Finally, adequate perfusion fixation is an absolute necessity, especially because the brain is extremely sensitive to postmortem changes and because these changes closely resemble lesions induced by mild hypoxia." In summary, restriction of maternal uterine blood flow resulted in increased fetal serum lactate concentra-
December 1993 Am J Obstet Gynecol
tions and severe asphyxia. During asphyxia animals increased their cerebral blood flow and remained normotensive and normoglycemic. Cerebrospinal fluid concentrations of lactate, hypoxanthine, and xanthine increased fivefold, fivefold, and fourfold, respectively. Maximum concentrations of hypoxanthine and xanthine were reached in the recovery period after asphyxia and were associated with edematous cell changes. In the recovery period the arterial oxygen content returned to baseline values. This reoxygenation may facilitate oxygen free radical formation and may thus aggravate postasphyctic brain damage. In distinguishing between histologic damage as a result of asphyxia or as a postmortem artifact, perfusion fixation appeared to be important. The absence of extensive histologic brain damage indicates that, under normotensive and normoglycemic conditions, the fetal brain can tolerate severe asphyxia. Cerebellar Purkinje cells tended to be most sensitive to asphyxia. We thank May Bost, Joyce Suyk, Jan Geilen, and Marc Haseldonckx for skillful technical assistance; Anke IJ zermans for assistance in performing the experiments; Frans Slangen and Peter Franssen for animal care; and Guy .Jacobs for preparation of the photographs. REFERENCES 1. Parer JT, Livingston EG. What is fetal distress? AM J OBSTET GVNECOL 1990;162:1421-7. 2. Low JA, Galbraith RS, Muir DW, Killen HL, Pater EA, Karchmar EJ. Motor and cognitive deficits after intrapartum asphyxia in the mature fetus. AM J OBSTET GVNECOL 1988; 158:356-61. 3. Goodwin TM, Belai I, Hernandez P, Durand M, Paul RH. Asphyxial complications in the term newborn with severe umbilical acidemia. AMJ OBSTET GYNECOL 1992;167:150612. 4. Myers RE. Two patterns of perinatal brain damage and their conditions of occurrence. AM J OBSTET GVNECOL 1972; 112:246-76. 5. Myers RE. Four patterns of perinatal brain damage and their conditions of occurrence in primates. Adv Neural 1975; 10:223-34. 6. Levine S. Anoxic-ischemic encephalopathy in rats. Am J PathoI1960;36:1-17. 7. Ting P, Yamaguchi S, Bacher JD, Killens RH, Myers RE. Hypoxic-ischemic cerebral necrosis in midgestational sheep fetuses: physiopathologic correlations. Exp Neural 1983;80:227-45. 8. Williams CE, Gunn AJ, Synek B, Gluckman PD. Delayed seizures occurring with hypoxic-ischemic encephalopathy in the fetal sheep. Pediatr Res 1990;27:561-5. 9. Saugstad OD. Hypoxanthine as an indicator of hypoxia: its role in health and disease through free radical production. Pediatr Res 1988;23: 143-50. 10. Van Belle H, Goossens F, Wynants J. Formation and release of purine catabolites during hypoperfusion, anoxia, and ischemia. Am J Physiol 1987;252:H886-93. 11. de Haan HH, de Haan J, Van Reempts JLH, Van Belle H, Hasaart THM. The effect of adenosine transport inhibition on cardiovascular function and survival after severe asphyxia in fetal lambs. Pediatr Res 1993;33:185-9. 12. Gluckman PD, Parsons Y. Stereotaxic neurosurgery on the ovine fetus. In: Nathanielsz PW, ed. Animal models in fetal
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