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ORIGINAL RESEARCH |
From the Department of Obstetrics and Gynecology, School of Medicine, Fukushima Medical University, Fukushima, Japan.
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ABSTRACT |
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 ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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METHODS: Regional blood flow was measured by the colored microsphere technique during continuous infusion (60 minutes) of big endothelin-1, or saline (control), in 12 chronically instrumented sheep fetuses.
RESULTS: After 60 minutes of big endothelin-1 administration, the fetal plasma endothelin-1 concentration increased significantly from 24.0 ± 6.7 to 49.7 ± 31.4 pg/mL (P = .018) without significant changes in fetal arterial blood gases. Continuous infusion of big endothelin-1 decreased blood flow in most organs except the brain and the heart. After the big endothelin-1 infusion, the blood flow to the brain significantly increased from 158 ± 51 to 174 ± 71 mL/min/100 g (P = .002); the blood flow to the heart also increased significantly from 171 ± 95 to 200 ± 112 mL/min/100 g (P = .001), respectively.
CONCLUSION: Continuous infusion of endothelin-1 decreases blood flow in most of organs except the brain and the heart. It is likely that endothelin-1 plays an important role in fetal redistribution of blood flow.
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MATERIALS AND METHODS |
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ABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Surgery was performed at 124 to 128 days of gestation (full term = 145 days) after a 24-hour maternal fast. Anesthesia was induced with atropine sulfate (1 mg) and xylazine (0.2 mg/kg) intramuscularly, and was maintained with ketamine (2 mg/kg) intravenously at 20-minute intervals. Using aseptic technique, a midline skin incision was made. The fetal head was delivered through a hysterotomy incision and covered with a surgical glove filled with warm saline. Polyvinyl catheters (Imamura Co., Tokyo, Japan, 1.7 mm outside diameter and 0.9 mm inside diameter) were inserted into the fetal carotid artery, jugular vein, amniotic cavity, and maternal femoral vein. Polyvinyl catheters were also placed in the fetal brachial and femoral artery (for the withdrawal of reference samples during microsphere injections), as well as the fetal brachial and femoral vein (for microsphere injections). Electrodes attached to polyvinyl-coated stainless steel wires (Cooner, Chatsworth, CA.) were placed on the fetal trunk to record fetal heart rate (FHR). Fetal catheters and electrodes and maternal catheters were exposed through an incision in the left flank of the maternal sheep. Fetal blood pressure was measured using a pressure transducer (Disposable Transducer Kit, Model DT-NN; Spectramed Medical Products Pte. Ltd., Singapore) and recorded continuously on a chart recorder (PowerLab System, AD Instruments Co., Colorado Springs, CO). The mean fetal blood pressure, corrected for intra-amniotic pressure, and FHR were analyzed every 10 minutes before blood samplings.
After surgery and before initiating the experiments, 1 g of ceftizoxime sodium was administered to the maternal sheep through the femoral vein catheter every 12 hours. The animals were allowed to recover for at least 48 hours postoperatively. Experiments were performed on the 12 fetuses at 126 to 130 days of gestation.
Nonradioactive8 colored polystyrene microspheres of 15 µm in diameter (E-Z Trac; Interactive Medical Technologies, Los Angeles, CA) were used. Vials obtained from the manufacturer contained approximately 5 x 106 microspheres per milliliter. The microsphere suspension contains thimerosal (0.01%), a bacteriostat, and Tween 80 (0.05%) hydrophobic spheres to prevent aggregation. Because the total number of microspheres needed for fetal sheep is 2 to 3 x 106, the total volume required for injection was 1 mL. In this study, yellow- and red-colored microspheres were used. Vigorous vortex agitating of the stock vial was performed for 1 minute immediately before the aliquot was withdrawn. The microspheres were drawn into a 3-mL syringe with an 18-gauge needle. The syringe was continuously agitated until the injection.
We obtained fetal baseline plasma endothelin-1 levels and blood gases at preinfusion period. After the preinfusion observations, we began infusing a big endothelin-1 (Funakoshi Co., Japan) with saline solution into the fetal jugular vein catheter (endothelin-1 infusion). A loading dose of 5 ng/kg of estimated fetal body weight was infused for 60 minutes (0.2 mL/min). In the controls, we conducted the same procedure as the endothelin-1 infusion with equivalent amounts of saline solution (control infusion).
Fetal blood was anaerobically drawn from the fetal carotid artery for the measurement of endothelin-1 and blood gases during the preinfusion period and 60 minutes after the onset of infusion. Blood gases and pH were measured using a blood gas analyzer (ALB 555; Radiometer Co. Copenhagen, Denmark) with the temperature corrected to 39°C. An additional 4.0 mL of arterial blood was drawn into a syringe treated with aprotinin and ethylenediaminetraacetic acid for analysis of endothelin-1. This blood sample was immediately centrifuged at 3,000 rpm for 15 minutes at 4°C. The plasma was separated and stored at –80°C until analysis. The plasma endothelin-1 concentrations were measured using a modified radioimmunoassay.9 The sensitivity of the endothelin-1 assay was 0.3 pg/mL. The interassay coefficient of variation for endothelin-1 was 10%, whereas the intra-assay coefficient of variation was 14.1%.
For regional blood flow measurement, syringes with 18-gauge needles containing the microspheres were attached to the brachial and the femoral vein, and the injections (containing 1.5 x 106 microspheres) were administered simultaneously over a 10-second interval into both veins. The catheters were then flushed with 3 mL of normal saline solution.
For a reference blood sample to calculate regional blood flow, the brachial (for upper organs reference) and the femoral artery (for lower organs reference) catheters were attached to heparin-filled 10-mL glass syringes placed on a precalibrated syringe pump (Harvard Apparatus, Dover, Mass). The syringe pump was set at 3.0 mL/min, resulting in a total withdrawal volume of approximately 6 mL over the course of the microsphere injections.
After the experiment, the ewe was killed with an overdose of sodium thiopental and potassium chloride; subsequently, the fetus was dissected. Representative portions of the fetal organs (1 to 2 g) were removed and blotted, placed into preweighed conical 15-mL polypropylene tubes, weighed, and assayed with the colored microsphere technique described by Hakkinen et al.10 The required samples are listed in Box 1.
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The tubed reference blood10 samples and removed fetal organs were digested with 2N sodium hydroxide. The microspheres were isolated using centrifugation, and the dye was extracted with dimethylformamide. The dye was quantified by spectrophotometry (Spectrophotometer U-2001, Hitachi Co., Tokyo, Japan). The absorption spectrum peaks for the yellow and red dyes are at 448 nm and 530 nm, respectively. Ten nanometer bandwidth filters, centered at 450 nm and 530 nm, were used to quantify dye extracted from the yellow and red microspheres, respectively. The absorbances at the yellow and red wavelengths for overlap from the red and yellow dyes, respectively, were corrected using spectral stripping.
The flow rate of the10 reference sample, in milliliters per minute, was calculated by dividing the reference blood volume by the time required for the withdrawal. For each infusion, the quotient of the reference blood flow rate divided by the activity of the reference sample was used as the basis for calculating tissue flow rate: Qs = As(Qr/Ar), where Qr represents the reference blood flow, Qs represents sample tissue blood flow, Ar represents the activity in absorbance units in the reference blood, and As represents the activity in absorbance units in the sample tissue. Blood flow rate was divided by tissue weight to yield milliliters per minute per 100 g.
All data are presented as a mean plus or minus one standard error of the mean. The Wilcoxon rank-sum test was used to evaluate differences in gestational ages. Statistical analysis was performed by means of the Wilcoxon signed-rank test to compare the postinfusion values with the preinfusion control values. The statistical tests were performed using the SPSS 12.0 program (SPSS Co., Tokyo, Japan). A value of P < .05 was considered statistically significant.
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RESULTS |
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In the control (Table 1) infusion group, fetal plasma endothelin-1 concentrations did not significantly alter during the observation period. Continuous infusion of a big endothelin-1 significantly increased the fetal plasma endothelin-1 concentration from 24.0 ± 6.7 to 49.7 ± 31.4 pg/mL at 60 minutes (P = .018) in the big endothelin-1 infusion group.
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Fetal arterial blood gases and pH did not significantly alter throughout the experiments in the control infusion group and the endothelin-1 infusion group. During the study period (Table 1), no significant change was found in the fetal blood pressure of either the control infusion group or the endothelin-1 infusion group. Furthermore, a significant change in the FHR was found in the endothelin-1infusion group (P = .018) but not in the control infusion group.
Regional blood flow data during experiments is presented in Table 2. In the control group, regional blood flow in all fetal organs did not significantly change between preinfusion and postinfusion.
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In the endothelin-1 infusion group, blood flow to the brain (P = .002) and heart (P = .001) significantly increased with the big endothelin-1 infusion. Blood flow to the lung (P = .03), liver (P = .018), kidneys (P = .001), intestines (P = .019) and placenta (P = .043) significantly decreased with the big endothelin-1 infusions. In the spleen (P = .237) and adrenal glands (P = .091), blood flow decreased; however, the decrease was not statistically significant. In examining specific brain regions in the endothelin-1 infusion group, blood flow to the cerebrum did not significantly change (P = .088); however, flow to the brainstem significantly increased (P = .009) from the preinfusion rate. Moreover, in examining specific cardiac areas for the endothelin-1 infusion group, blood flow to the atria did not significantly change (P = .245); however, flow to the ventricles significantly increased (P = .004) from the preinfusion rate.
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DISCUSSION |
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Endothelin-1, a 21 amino acid polypeptide, produced by vascular endothelial cells, has potent vasoactive properties.13 Endothelin-1 is initially synthesized as a 203-amino acid prepropeptide (prepro endothelin-1), which is the cleaved by an endopeptidase to proendothelin-1 (big endothelin-1). A big endothelin-1, 38-amino peptide, is then converted to endothelin-1 by an endothelin-converting enzyme, which is a membrane-bound metalloprotease present in endothelial and smooth muscle cells.14 Endothelin-1 is a potent vasoconstrictor peptide that probably has an important role in the maintenance of basal vasomotor tone, interacting with other vasoactive agents, such as catecholamines, potentiating their vasoactive effect.15
The hemodynamic effects of endothelin-1 are mediated by at least 2 distinct receptor populations, ETA and ETB, the densities of which vary depending on the vascular bed studied. The ETA receptors are located on vascular smooth muscle cells and mediate vasoconstriction, whereas the predominant subpopulation of ETB receptors are located on endothelial cells and mediates vasodilation. However, a second subpopulation of ETB receptors is located on smooth muscle cells and mediates vasoconstriction.19 The vasodilating effects of endothelin-1 are associated with release of NO and potassium channel activation. The vasoconstricting effects of endothelin-1 are associated with phospholipase activation, the hydrolysis of phosphoinositol to inositol-1,4,5-triphosphate and diacylglycerol, and the subsequent release of Ca2+. In addition to its vasoactive properties, endothelin-1 has mitogenic activity on vascular smooth muscle cells and may participate in vascular remodeling.
The predominant effect of exogenous endothelin-1 in the fetal and newborn sheep pulmonary circulation is vasodilation, mediated via ETB receptor activation and NO release. However, the predominant effect in the juvenile and adult pulmonary circulation is vasoconstriction, mediated by ETA receptor activation. In fetal lambs, a selective ETA receptor blockade produces small decreases in resting fetal pulmonary vascular resistance.20,21 This suggests a potential minor role for basal endothelin-1–induced vasoconstriction in maintaining a high fetal pulmonary vascular resistance. Moreover, Green et al22 studied the response of endogenous endothelin-1 by ETA receptors antagonist during normoxemic and hypoxemic condition in ovine fetuses. They reported that the ETA receptors antagonist created the increase in carotid blood flow during normoxemia and the decrease in femoral blood flow during acute hypoxemia. In the renal circulation of fetal sheep, endothelin-1 in vivo has a vasodilatory effect. Endothelin-1 was shown to act primarily through ETB receptors, producing vasodilation; however, ETA receptors, mediating vasoconstriction were shown to contribute to renal vascular tone.23 Docherty et al24 reported that the responses to endothelin-1 of isolated adrenal, femoral, middle cerebral, and renal arteries from fetal (110–145 days of gestation) and newborn sheep were evaluated by using the technique of wire myography in vitro. Sensitivity to endothelin-1 increased with increasing fetal age in arteries from all vascular beds. They also suggested that the augmented sensitivity to endothelin-1 of the middle cerebral when compared with other systemic arteries might reflect the importance of cerebral blood flow control during the critical developmental period. These data supported our observations that exogenous endothelin-1 could create a fetal redistribution effect in fetal sheep; however, blood flow to the adrenal glands did not increase after exogenous endothelin-1 administration when compared with other animal studies.2,3 It seems that the fetal redistribution, which occurs under hostile conditions, might be due not only to endothelin-1 but also to other vasoactive substances. Further observations are needed to clarify the stimuli for fetal redistribution.
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Footnotes |
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doi:10.1097/01.AOG.0000178764.35532.18
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REFERENCES |
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20. Ivy DD, Kinsella JP, Abman SH. Physiologic characterization of endothelin A and B receptor activity in ovine fetal pulmonary circulation. J Clin Invest 1994;93:2141–8.
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