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Endocrinology, doi:10.1210/en.2006-1058
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Endocrinology Vol. 148, No. 3 1288-1295
Copyright © 2007 by The Endocrine Society

The Effects of Maternal Betamethasone Administration on the Intrauterine Growth-Restricted Fetus

Suzanne L. Miller, Mahalia Chai, Jan Loose, Margie Castillo-Meléndez, David W. Walker, Graham Jenkin and Euan M. Wallace

Departments of Physiology (S.L.M., M.C., J.L., M.C.-M., D.W.W., G.J.) and Obstetrics and Gynaecology (E.M.W.), Monash Immunology and Stem Cell Laboratories (G.J.), Monash University, Clayton, Victoria 3800, Australia

Address all correspondence and requests for reprints to: Prof. Euan M. Wallace, Centre for Women’s Health Research, Department of Obstetrics and Gynecology, Monash Institute of Medical Research, Monash Medical Centre, 246 Clayton Road, Clayton, Victoria 3168, Australia. E-mail: euan.Wallace{at}med.monash.edu.au.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Intrauterine growth restriction (IUGR) is associated with altered fetal cardiovascular function to ensure adequate perfusion of essential organs. IUGR fetuses are at risk of preterm delivery and so are likely to receive antenatal glucocorticoids to promote lung maturation. Because glucocorticoids alter vascular tone, we questioned whether such treatment may induce fetal cardiovascular alterations. Using pregnant sheep carrying twins, we induced IUGR at approximately 0.7 gestation by single umbilical artery ligation in one twin, using the other twin as a control. In each fetus, we monitored carotid blood flow and arterial blood gases. We administered 11.4 mg betamethasone (n = 5) or vehicle (n = 4) to the ewe on d 5 (BM1) and 6 (BM2) postsurgery. On d 7, fetal brains were collected for immunohistochemistry. In control fetuses, carotid blood flow decreased 3.5 h post-BM1 by 24% (P < 0.001), returning to baseline at 5.5 h. In IUGR fetuses, carotid flow decreased 2.5 h post-BM1 by 27% and then increased by 25% over baseline, peaking at 11 h (P < 0.001). Compared to control + saline, we observed a significant increase in oxidative damage (4-hydroxynonenal-positive cells) in the fetal hippocampus and subcallosal area of all treatment groups (IUGR + BM > IUGR + saline = control + BM). There was a significant correlation between carotid blood flow reperfusion after betamethasone and the number of 4-hydroxynonenal-positive cells in the cortex and hippocampus. These data suggest that antenatal betamethasone may induce brain injury in the IUGR fetus but not in the normally grown fetus.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN PREGNANCIES COMPLICATED by severe intrauterine fetal growth restriction (IUGR) and absent end-diastolic flow in the umbilical artery Doppler flow velocity waveform, the administration of the synthetic glucocorticoid betamethasone to the mother in preparation for preterm delivery is associated with a transient return of end diastolic umbilical artery flow (1, 2, 3), and similar changes in Doppler flow velocity waveforms in other fetal vascular beds (4). Although these Doppler changes indicate that betamethasone has potentially important cardiovascular effects in the IUGR fetus, most likely a generalized transient vasodilation, whether these changes are beneficial or detrimental to the IUGR fetus is not known (3). Of note, the changes do not occur when normally grown fetuses are exposed to betamethasone (4, 5)

Consideration of the potential cerebral effects of these synthetic glucocorticoid-induced changes in the circulation is important because there is both experimental and clinical evidence that the brain of the IUGR fetus is particularly at risk of damage. In ovine models of IUGR, induced by chronic placental insufficiency, the fetuses sustain significant neuropathology including cortical gliosis, decreased myelination of the subcortical white matter, and reduced numbers of Purkinje neurons in the cerebellum. Cerebral capillary diameter is also increased, indicative of prolonged vasodilation (6, 7). Similarly, in humans, severe IUGR is associated with ischemic cerebral injury, permanent neurological damage, and reduced cognitive function of school-age children (8, 9). Because the IUGR fetus has a higher risk of requiring preterm delivery (10), it is likely to receive antenatal glucocorticoids in preparation for birth. What effect this might have on cerebral blood flow and function has not been studied previously.

Glucocorticoids are powerful regulators of vascular function, and a number of studies have shown that synthetic glucocorticoids, such as betamethasone, alter fetal cardiovascular parameters in otherwise normal pregnancies. In healthy fetal sheep, late-gestation maternal glucocorticoid administration results in fetal hypertension (11, 12) accompanied by increased vascular resistance (11, 13), mild hypoxemia (14), and decreased cerebral blood flow (15). Synthetic glucocorticoids also have profound effects on the developing brain. Normally grown sheep fetuses in late gestation exposed to maternal glucocorticoids show reduced brain weight (16), a delay in myelination (17), a reduction in astrocyte density (18), and altered neuronal cytoskeleton (15). Furthermore, the reduced brain weight evident at term in the fetal sheep that received antenatal steroids (15) is also present in adulthood (19). There is also evidence of glucocorticoid-induced CNS damage in primate models, where antenatal glucocorticoids decrease numbers of pyramidal neurons (20, 21). Some of these effects arise from direct damage on brain cell development, but some adverse effects may be due to altered cerebrovascular regulation after treatment. For example, if the vasodilator effects of betamethasone results in redirection of blood flow away from the brain, then it is possible that such treatment could further compromise the cerebrovascular function and brain structure in IUGR fetuses.

It is clear that IUGR or synthetic glucocorticoid administration individually have profound acute and long lasting effects on cardiac and neurological development and function. The aim of the current study was to investigate whether, in combination, IUGR and antenatal glucocorticoids have additional effects on fetal blood flow and on markers of brain injury.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal care and surgical preparation
All procedures were conducted with the approval of the Physiology Animal Ethics Committee, Monash University. Surgery was performed on 10 twin-bearing Border-Leicester Merino crossbred ewes with fetal gestational ages between 105 and 110 d (term ~147 d). Anesthesia was induced with 20 mg/kg sodium thiopentone (Pentothal; Bomac Laboratories Ltd., Asquith, Australia) in 5 ml sterile water; iv. General anesthesia was maintained with 2.5% isoflurane in 100% oxygen (ISOFLO; Abbott Australasia Pty. Ltd., Kurnell, Australia). Under aseptic conditions, the uterus was exposed through midline incision on the maternal abdomen. In each fetal twin, a sterile polyvinyl catheter [1.5 mm outer diameter (OD), 0.8 mm inner diameter (ID)] was inserted into the fetal femoral artery and a ultrasonic flowprobe (size 3; Transonic Systems, Ithaca, NY) was placed around the right carotid artery. A catheter (4.8 mm OD, 2.6 mm ID) was also placed into each amniotic cavity. Catheters were filled with sterile heparinized saline (0.9% NaCl: 25,000 IU heparin/liter; Baxter, New South Wales, Australia). The umbilical arteries were identified within the cord approximately 3 cm from the fetal abdomen, and single umbilical artery ligation (SUAL) was undertaken in one twin by placing two ligatures around one of the umbilical arteries. The cord was manipulated but not ligated in the control twin. The fetus was then returned to the uterus and the catheters and flow probes were exteriorized via an incision in the flank of the ewe. A three-wire electromyography lead was sutured to the external wall of the uterus to detect signs of labor. A catheter (1.5 mm ID x 2.7 mm OD) was also inserted into the maternal jugular vein. After surgery, the ewes were housed in metabolic cages and fed once daily. Water was provided ad libitum.

All ewes received ampicillin (1000 mg in 5 ml heparinized saline, Austrapen, CSL Ltd., Parkville, Victoria, Australia) at the time of surgery and catheters were flushed daily with sterile heparinized saline to ensure patency.

Experimental protocol
All experiments commenced after a 4-d recovery period after surgery. On d 4, we began continuous monitoring of carotid blood flow; fetal mean arterial pressure (MAP) was electronically corrected for changes of amniotic fluid pressure and heart rate (HR) using a PowerLab (ADInstruments, Castle Hill, New South Wales, Australia). On d 5, ewes received 11.4 mg im betamethasone (Celestone Chronodose; Schering Plough, Baulkham Hills, Australia), termed BM1, and a second 11.4 mg betamethasone administration 24 h later (BM2). The dose of betamethasone was chosen to reflect the dose used clinically in preparation for preterm birth (1, 2, 4). In both human and ovine fetuses, the effects of betamethasone are considered to be exerted principally via glucocorticoid receptor mediated events (22), consistent with the original observation that dexamethasone administration to ovine fetuses mimicked the effects of fetal infusions of either ACTH or cortisol (23). Control ewes received an equal volume of im saline. Fetal arterial blood samples were collected at 24 h and 1 h before each betamethasone dose and at 2, 6, 8 and 12 h after each dose. These samples were immediately analyzed for blood gases (PaO2, PaCO2, SaO2), hemoglobin concentration, and pH using an ABL5 acid-base blood gas analyzer (Radiometer, Copenhagen, Denmark). On d 7 (24 h after BM2), the ewe was killed with an overdose of pentobarbitone (Lethabard Virbac Pty. Ltd., Peakhurst, New South Wales, Australia) and the fetuses were weighed. The fetal brains were perfused in situ with approximately 1 liter 0.9% saline solution (Baxter) via the common carotid artery, and then fixed by perfusion of 1 liter 4% buffered paraformaldehyde (Prosci Technology, Thuringowa, Queensland, Australia) in 0.1 M phosphate buffer (pH 7.4). The brains were postfixed by immersion in 4% paraformaldehyde for 24 h and then processed for paraffin embedding.

Immunohistochemistry
4-Hydroxynonenal (4HNE) and caspase-3 staining were performed on paraffin-embedded coronal sections (10 µm) mounted on Superfrost Plus slides (Menzel-Glaser, Braun-Schweig, Germany) as previously described (24). Briefly, sections were deparaffinized in xylene, rehydrated in descending alcohol concentrations (2 x 100% and 70%, 5 min each) and then in distilled water, and washed in PBS three times for 5 min. Antigen retrieval was undertaken in citric buffer (0.01 M, pH 6; ICN Biomedicals Inc., Irvine, CA) using three 2-min bursts in the microwave and allowing the sections to cool in buffer. After washes (three times for 5 min) in PBS containing 0.3% Triton-X (Sigma-Aldrich, St. Louis, MO), endogenous peroxidase activity was blocked by incubating tissue sections in 3% hydrogen peroxide (BHD Analar, Poole, UK) in 50% methanol for 30 min. Sections were preblocked with 5% normal goat serum (IMVS Veterinary Services, Adelaide, South Australia, Australia) for 45 min at room temperature, and incubated overnight at 4 C in a 1:100 dilution of a rabbit antihuman/mouse activated caspase-3 primary antibody (R&D Systems, Minneapolis, MN) or 4HNE rabbit polyclonal antibody (1:200; Calbiochem, San Diego, CA). After washing in PBS, sections were incubated in 1:200 dilution of a biotinylated goat antirabbit secondary antibody (Vector Laboratories, Burlingame, CA) for 1 h. Immunoreactivity was visualized by the addition of metal enhanced diaminobenzidine (Pierce, Rockford, IL; 1:10). Further reaction was stopped by washing sections in distilled water. The sections were dehydrated in ascending alcohols, cleared with xylene, and coverslipped. Negative controls that omitted the primary antibody were included in each run.

Sections were imaged with a Motic 200 camera and Motic Vision computer software (Australian Instrument Services, Bayswater, Australia) under light microscopy (x400 magnification; Zeiss Axiocam, Thornwood, NY). Cells were counted in predetermined areas in the parasagittal cortex, subcallosal bundle, caudate nucleus, and the hippocampus. The number of immunopositive cells in each region was calculated using the average of four fields of vision per region. Two slides per fetus for each brain region were photographed and counted. Quantification was performed by two independent counters blinded to the treatment group.

Data analysis
Data are presented as mean ± SEM. Data analysis was performed with SPSS statistical software (SPSS 11.5 software package for Windows). Differences in fetal weight, brain weight, and brain to body weight ratio were examined using one-way ANOVA. Serial measurements of carotid blood flow, MAP, HR, blood gases, and pH were analyzed by two-way repeated measures ANOVA followed by least significant difference applied post hoc. The number of immunopositive cells were analyzed by two-way repeated measures ANOVA, with post hoc least significant difference when required. Analysis of continuous data (i.e. carotid blood flow, MAP, and HR) was undertaken by taking a mean of the data over 1 min, every 30 min. The change in carotid flow was calculated by standardizing the flow at time –1 h to a value of 1 and referencing all other points against this value. Correlations between reperfusion ratio and immunopositive cell counts were analyzed using the Spearman test. Statistical significance was accepted when P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
One ewe showed signs of advanced labor within 24 h of betamethasone administration and, therefore, has not been included in the results, which subsequently represent n = 5 for betamethasone administration, control + BM, and IUGR + BM; and n = 4 for saline treatment, control + saline, and IUGR + saline.

Body and brain weights
At the time of postmortem, the body weight of fetuses in the IUGR + saline, control + BM, and IUGR + BM treatment groups were all significantly decreased (F[3,16] = 12.84, P < 0.001) compared with the control + saline group (Table 1Go). The absolute brain weights did not differ between groups but, compared with control + saline, the brain to body weight ratio was significantly increased in IUGR + BM fetuses (F[3,16] = 3.22, P = 0.05) (Table 1Go).


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  		TABLE 1. Mean ± SEM outcomes for ovine twin fetuses with/without single umbilical artery ligation receiving saline or betamethasone

 
Fetal arterial blood gases and pH
Before administration of BM, fetal arterial O2 saturation was significantly lower in the IUGR fetuses compared with their control twins (58.9 ± 2.8% vs. 67.7 ± 2.1%) (F[1,30] = 6.07, P = 0.02; Fig. 1Go). Overall, there was a trend for O2 saturation levels to fall after betamethasone and, compared with levels before treatment, they was significantly decreased in the control + BM fetuses at 24 h after BM2 (F[10,30] = 6.735, P < 0.001) and in IUGR + BM fetuses at 12 and 24 h after BM2 (F[10,30] = 2.22, P < 0.05; Fig. 1Go), when compared with basal levels before treatment. Before BM, there were no differences between control and IUGR fetuses with regard to fetal arterial pO2 (20.02 ± 0.67 vs. 18.06 ± 0.78 mm Hg) (F[1,30] = 3.63, P = 0.06), pCO2 (43.71 ± 0.98 vs. 43.26 ± 0.88 mm Hg) (F[1,30] = 0.11, P = 0.74) or pH (7.37 ± 0.004 vs. 7.35 ± 0.009) (F[1,30] = 3.05, P = 0.09), nor were there any changes in these parameters over time in response to BM administration.


Figure 1
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  		FIG. 1. Fetal arterial oxygen saturation in response to 11.4 mg betamethasone (BM1) or saline into the ewe, with a second administration after 24 h (BM2). {dagger}, A significant difference in the control + BM group at BM2 + 24 h, compared with basal (P < 0.001); *, a significant difference in the IUGR + BM group at BM2 + 12 h and + 24 h compared with basal (P < 0.05).

 
Fetal MAP and HR
Before treatment, there was no difference in fetal MAP between control and IUGR fetuses (34.7 ± 2.5 vs. 35.5 ± 1.5 mm Hg) (F[1,12] = 0.08, P = 0.79). After BM1 administration, fetal MAP was significantly increased in the control + BM group at 1.5 h (40.7 ± 1.0 mm Hg) (F[20,80] = 4.62, P < 0.001) compared with pretreatment levels and remained elevated for the duration of the experiment. Similarly, in the IUGR + BM fetuses, MAP was significantly elevated at 2.5 h after BM1 administration (43.6 ± 2.3 mm Hg) (F[20,80] = 4.07, P < 0.001) and thereafter remained elevated. There was no difference in the fetal HR between control and IUGR fetuses before treatment (178 ± 3 vs. 169 ± 7 beats/min) (F[1,12] = 1.28, P = 0.028) and in response to BM1, there was no change in control HR, but in IUGR fetuses, HR was significantly increased at 5.5–6.5, 7.5, and 9 h, to a maximum of 182 ± 6 beats/min at 7.5 h (F[20,60] = 4.11, P < 0.001).

Fetal carotid blood flow
Before BM or saline administration, there was a significant difference in basal carotid blood flow between the experimental groups, with flows ranging from 25.2 ± 1.8 ml/min in the IUGR + BM fetuses to 41.4 ± 2.1 ml/min in the control + saline group (F[4,16] = 5.07, P = 0.01). Due to this difference, all carotid blood flow values were expressed as change in flow relative to baseline. Using two-way ANOVA, there was a significant interaction between time and group for change in blood flow (F[60,240] = 1.505, P = 0.02) and, therefore, individual treatment groups were analyzed. There was no change in carotid blood flow in the control + saline group (F[60,120] = 1.21, P = 0.19); however, in the IUGR + saline group, carotid blood flow was increased at 12.5 and 14 h after saline (F[60,120] = 1.63, P = 0.012) compared with basal levels (data not shown). In the control + BM fetuses, after BM1, carotid blood flow was significantly decreased between 3.5 and 5 h, with a maximum decrease of 24 and 9% at 5 h (Fig. 2Go; F[60,240] = 2.41, P < 0.001) when compared with blood flow pretreatment. There was no significant change in carotid flow in response to BM2. The IUGR + BM fetuses demonstrated a biphasic change in carotid blood flow in response to BM1, with a significant decrease between 2.5 and 6.5 h after BM1 (maximum decrease of 27 ± 4% at 5 h) when compared with basal values, and then a significant increase in carotid blood flow between 9.5 and 13.5 h after BM1, with a maximum increase of 25 ± 11% at 11 h (F[60,240] = 5.46, P < 0.001). In response to BM2, there was no significant change in carotid blood flow in IUGR + BM fetuses (Fig. 2Go). A reperfusion ratio was calculated in BM-treated animals to determine the degree of the rebound in different animals. The calculation was performed by dividing the maximum carotid flow (reperfusion) in response to BM by the minimum carotid flow, for each animal. The mean (range) reperfusion ratio in IUGR + BM fetuses was 1.91 (1.68–2.28), significantly greater than the ratio in control + BM fetuses: 1.5 (1.35–1.85), (F[1,8] = 8.73, P = 0.02).


Figure 2
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  		FIG. 2. Carotid blood flow, shown as change from control, in control + betamethasone fetuses (bullet) and IUGR + betamethasone fetuses ({circ}) in response to 11.4 mg betamethasone, into the ewe at BM1 and BM2. *, P < 0.05 denotes significant differences in control + BM and IUGR + BM compared with respective prebetamethasone values; **, P < 0.05 denotes a significant difference in IUGR + BM compared with prebetamethasone; {dagger}, P < 0.05 represents a significant difference in control + BM vs. IUGR + BM.

 
Immunohistochemistry
The level of lipid peroxidation, as shown by 4HNE immunoreactivity, was low throughout the brain in the control + saline fetuses in all regions examined (Fig. 3AGo). There was a significant interaction between brain area and treatment group for the analysis of 4HNE immunoreactivity (F[9,48] = 2.80, P = 0.01) and, therefore, individual brain areas were examined with one-way ANOVA. In the cortex, IUGR + BM animals demonstrated significantly increased numbers of 4HNE-stained cells when compared with control + saline (F[3,16] = 3.18, P = 0.05). Within the hippocampus, betamethasone treatment alone (control + BM fetuses), IUGR alone (IUGR + saline fetuses), or combined treatments (IUGR + BM) resulted in a significant increase in the number of 4HNE immunopositive cells (F[3,16] = 10.95, P < 0.05). In addition, the number of cells positive for 4HNE was significantly increased in IUGR + BM fetuses when compared with IUGR + saline (P = 0.015) and control + BM (P = 0.002) in the hippocampus (Fig. 4Go), indicative of an additive or augmenting effect of BM treatment in IUGR fetuses. In the subcallosal region, all treatment groups showed increased 4HNE immunoreactivity compared with control + saline (F[3,16] = 15.04, P < 0.05) and IUGR + BM demonstrated increased 4HNE immunoreactivity when compared with IUGR + saline (P < 0.001) and control + BM (P = 0.05). Within the caudate nucleus, there were no significant differences across groups for 4HNE immunostaining (F[3,16], = 2.92, P = 0.07).


Figure 3
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  		FIG. 3. Number of 4HNE (A)- and caspase-3 (B)-reactive cells per field of view in brain regions collected from control or IUGR fetuses exposed to BM or saline. *, P < 0.05 vs. control + saline; {dagger}, P < 0.05 vs. IUGR + saline and control + BM; **, P < 0.05 vs. cortex, subcallosal, and caudate nucleus.

 

Figure 4
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  		FIG. 4. Photomicrographs showing representative 4HNE immunoreactivity in the CA1 region of the hippocampus of control + saline fetus (A), IUGR + saline fetus (B), control + betamethasone fetus (C), and IUGR + betamethasone fetus (D). Scale bar, 100 µm.

 
Activated caspase-3 immunoreactivity was detectable in all brain regions examined in the control + saline fetuses (Fig. 3BGo), where caspase-3 immunoreactivity appeared to be nuclear. Two-way ANOVA revealed that there was a significant main effect for treatment group (F[3,16] = 3.53, P = 0.04), with all treatment groups, IUGR + saline, control + BM, and IUGR + BM, showing increased caspase-3 immunostaining compared with control + saline. There was also a statistically significant difference in the brain areas examined (F[3,9] = 10.99, P < 0.001), which was due to significantly increased activated caspase-3 immunoreactivity in the hippocampus compared with all other brain areas (Fig. 5Go). There was no interaction between treatment group and brain area for caspase-3 staining (F[9,48] = 0.56, P = 0.82).


Figure 5
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  		FIG. 5. Photomicrographs showing representative caspase-3 immunoreactivity in the CA1 region of the hippocampus of control + saline fetus (A), IUGR + saline fetus (B), control + betamethasone fetus (C), and IUGR + betamethasone fetus (D). Scale bar, 100 µm.

 
There was a significant correlation between the reperfusion ratio and the number of 4HNE-positive cells in the cortex (P = 0.006, r2 = 0.62), the hippocampus (P = 0.04, r2 = 0.43), and in all brain regions examined together (P = 0.017, r2 = 0.531) (Table 2Go). There was no apparent relationship between the reperfusion ratio and the number of activated caspase-3 cells in any brain areas examined.


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  		TABLE 2. Correlation between reperfusion ratio and 4HNE or activated caspase-3 immunostaining in various brain regions

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study extends our previous clinical observations that the fetal cardiovascular response to maternal betamethasone administration in IUGR fetuses is significantly different than that seen in healthy fetuses. Specifically, we have shown that the IUGR fetus displays rebound cerebral perfusion not seen in the healthy fetus, which is associated with increased oxidative stress and apoptosis in the fetal brain. This study provides the first experimental evidence that maternally administered glucocorticoids may have deleterious effects on the brain of IUGR fetuses.

IUGR was induced in fetal sheep at approximately 0.7 gestation by SUAL, using twin pregnancies to provide an internal age-matched control. SUAL alone (no betamethasone) resulted in a decrease in fetal body weight of almost 30% without an effect on brain weight, confirming our previous observation that SUAL induces asymmetric fetal growth restriction in singleton fetuses (25) similar to that seen in human IUGR. SUAL induces placental insufficiency by producing infarction of approximately half of the cotyledonary placentomes (26), thus reducing oxygen and substrate transfer. In this study, SUAL-induced IUGR fetuses were chronically hypoxemic with a significant reduction in fetal arterial oxygen saturation but were nonacidotic.

It is established that the administration of glucocorticoids is associated with significant vascular effects. In healthy sheep and primate fetuses, antenatal glucocorticoid administration induces hypertension (11, 12, 13, 27), an effect that was observed in this current study. Indeed, we have extended those previous studies by showing that the degree of hypertension induced by betamethasone was no different between IUGR and control fetuses. In human fetuses, we have previously shown that maternal administration of betamethasone in pregnancies complicated by fetal umbilical artery absent end-diastolic flow results in a transient return of end-diastolic flow in about two thirds of pregnancies (1, 2). This return of end-diastolic flow in the umbilical artery is accompanied by a decrease in the middle cerebral artery pulsatility index and other fetoplacental Doppler changes suggestive of widespread vasodilatation (4). These flow changes may interfere with the circulatory adaptations that are apparent in fetuses affected by IUGR including redistribution of cardiac output to maintain oxygen delivery to vital organs such as the brain (28). Indeed, the results obtained in the current study suggest that betamethasone administration is detrimental to the IUGR fetus with respect to fetal body weight, changes of carotid blood flow, and indices of brain injury.

An important and novel finding of this study is that the carotid blood flow response to betamethasone was distinctly different in the IUGR and control ovine fetuses. Initially, both groups of fetuses responded to betamethasone with a significant decrease in carotid blood flow, of a similar magnitude, at 2.5–3.5 h after treatment. Such a decrease in cerebral blood flow has been reported previously in healthy sheep fetuses after betamethasone (15). In the current study, 6.5 h after betamethasone carotid blood flow in both control and IUGR fetuses had returned to baseline. However, although the carotid blood flow in control fetuses remained stable, thereafter carotid blood flow in the IUGR fetuses demonstrated a large, significant rebound increase 9.5 h after betamethasone, persisting for 4 h. This finding is consistent with our clinical observation that cerebral Doppler flow velocity waveforms are significantly altered in IUGR fetuses after betamethasone administration but not in healthy fetuses (4). In the present ovine study, we chose to use carotid blood flow as an index of cerebral blood flow to obtain a continuous measure of the changes of global brain blood flow. Carotid blood flow provides a reliable and accurate prediction of flow to the total brain over a wide range of values (29) and has been used previously as a measure of global cerebral blood flow (30, 31). In view of our observations, it would now be worthwhile examining whether there are region-specific changes in blood flow within the brain, exploring whether regional differences were associated with patterns of brain injury.

Indeed, we believe that the rebound perfusion observed in the IUGR, but not control, fetuses is likely to be associated with increased risks of brain injury. The excessive reoxygenation that would be expected to occur during the rebound perfusion phase is likely to provide excess oxygen as a substrate for a range of enzymatic oxidative reactions and subsequently lead to the overproduction of reactive oxygen species (ROS) in mitochondria (32), leading to lipid peroxidation. The brain is particularly vulnerable to oxidative damage due to its high oxygen requirement, high lipid composition, and relatively low content of antioxidant enzymes (33, 34). In this study, IUGR alone, with no betamethasone, significantly increased cerebral lipid peroxidation, as assessed by 4HNE immunoreactivity, in the hippocampus and subcallosal area, consistent with a previous report of an association between IUGR and elevation of lipid peroxidation products in the amniotic fluid (35). Similarly, our observation in this study that healthy fetuses receiving betamethasone also demonstrated increased lipid peroxidation in the hippocampus and subcallosal bundle compared with saline-treated controls, concurs with a previous report of increased lipid peroxidation in the rat brain after dexamethasone administration (36). A potential mechanism for this increased peroxidation in healthy fetuses is the known suppression of the antioxidant enzyme glutathione peroxidase by glucocorticoids that has been observed in hippocampal cultures in vitro (37).

In this study, we have assessed for the first time the potential effects of glucocorticoids and fetal growth restriction—a likely combination in clinical practice—on lipid peroxidation. We have shown that the highest degree of lipid peroxidation in the hippocampus and subcallosal bundle was observed in the IUGR and betamethasone group, suggesting an additive, or augmenting effect of BM treatment in IUGR fetuses. It is not known how IUGR induces cerebral lipid peroxidation, but we have previously shown that severe acute hypoxia increases production of ROS and lipid peroxidation (25, 38), and it is possible that long-term moderate hypoxia may act in the same manner. The additional adverse effect of glucocorticoid treatment may be via a direct action on the cells (39) or may be mediated by the reperfusion in cerebral blood flow that we observed.

Lipid peroxidation and, specifically, the presence of 4HNE have been linked to cell death in the brain (40). In the current study, we observed increased apoptosis (activated caspase-3 immunoreactivity) across all treatment groups, compared with control fetuses treated with saline, and there was significantly more caspase-3 staining in the hippocampus compared with all other brain regions examined. We used an antibody specific for the activated form of caspase-3 that is only present in cells during apoptosis. Previous work has demonstrated that experimentally induced IUGR in fetal sheep results in significant brain pathology including a decrease in white matter myelination, Purkinje cell death, reactive astrocytosis, and cerebral vessel vasodilatation (5, 6). Similarly, glucocorticoid administration has been shown to reduce astrocyte density, delay myelination, and induce neuronal degeneration in developing sheep and primates (16, 17, 20). To date, our only assessment of cell death is using caspase-3, which is linked to the final events in the execution of the cell death program via specific and extensive DNA fragmentation (41). It is possible that, at 24 and 48 h after betamethasone administration, apoptotic activity has yet to reach a maximum and further study of cellular damage in this model is warranted given the changes in blood flow and lipid peroxidation. Furthermore, cell death may occur by nonapoptotic pathways. A recent study using a model of permanent brain ischemia in the adult rat has suggested that caspase-3 synthesis and activation is not essential for the execution of apoptosis and DNA fragmentation. The study showed two types of programmed cell death after brain ischemia: caspase-3-dependent cell death that peaked after 24 h of ischemia and caspase-3-independent apoptotic cell death that occurred after 48–72 h of ischemia (42).

To assess whether changes in blood flow were associated with peroxidative damage, we calculated a reperfusion ratio in response to betamethasone administration, defined as the ratio between the maximum postinsult flow value and the minimum value. The reperfusion ratio has been used previously to examine carotid blood flow changes after acute umbilical cord occlusion, showing a relationship between rebound perfusion and hippocampal neuronal damage (31). In our study, the carotid blood flow reperfusion ratio was significantly greater in the IUGR fetuses after betamethasone compared with the control fetuses, and overall we observed a significant correlation between the reperfusion ratio and immunoreactive 4HNE staining in the cortex and hippocampus. There was no such correlation between the reperfusion ratio and caspase-3 immunostaining. These data support the hypothesis that it is the reperfusion of the brain after betamethasone-induced reductions in perfusion in IUGR fetuses that induces the cerebral lipid peroxidation and that, in metabolically compromised cells, results in generation of excess free radicals (43).

In conclusion, in this study, we have shown that IUGR fetuses exposed to betamethasone are severely growth restricted, to a greater extent than IUGR alone. We have also shown that IUGR fetuses display a significant carotid blood flow reperfusion in response to maternal betamethasone that is not seen in healthy fetuses, and this rebound perfusion is correlated with lipid peroxidation within the fetal brain, which in turn contributes to an increased incidence of cell death. Our results demonstrate that the hippocampus and subcallosal area are particularly vulnerable to IUGR alone, betamethasone administration alone, and combined IUGR plus betamethasone treatments. These observations provide an insight into possible effects of betamethasone treatment on the IUGR human fetus that may have implications for the clinical management of the IUGR fetus, providing the impetus to investigate further therapeutic strategies for these babies.


   Footnotes
 
This work was undertaken with funding from a National Health and Medical Research Council (Australia) Program Grant (no. 334011), a National Health and Medical Research Council Career Development Award to E.M.W. (no. 194467), and by a Royal Australian and New Zealand College of Obstetricians and Gynaecologists Scholarship to S.M.

Disclosure Statement: The authors have nothing to declare.

First Published Online December 7, 2006

Abbreviations: BM, Betamethasone; 4HNE, 4-hydroxynonenal; HR, heart rate; ID, inner diameter; IUGR, intrauterine growth restriction; MAP, mean arterial pressure; OD, outer diameter.

Received August 2, 2006.

Accepted for publication November 28, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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