The human placenta has a dual fundamental role: It connects the embryonal, and later the fetal circulation to the maternal circulation, and it isolates the conceptus from the maternal organism. Fetal circulation in the trophoblast starts in the very early period of pregnancy: It can be detected from days 21–23 of gestation.1 Sufficient placental circulation is indispensable for the healthy development of the fetus, for an uncomplicated delivery, and for the tolerance of stress from birth.
An acute insufficiency of the placental circulation results in hypoxia, acute in utero distress, preterm birth, spontaneous abortion, or in utero death. A chronic insufficiency of the placental circulation can cause intra-uterine growth restriction,2 dysmaturity, and low birth weight. A severe placental insufficiency can cause fetal myocardial cell damage3 and coronary heart disease in adulthood.4 Clinical evidence has linked an intrauterine compromise, such as a prolonged period of insufficient placental circulation during the last trimester, to a poor neurological outcome in the newborn, which may manifest its consequences only at the ages of 5–8.5 At the present time, the mechanisms responsible for the regulation of placental blood flow are poorly understood. Moreover, detailed knowledge is needed of the direct effects of all the drugs that may be given during pregnancy and during delivery on the human placental vasculature. There have been several reports on the direct placental vascular effects of different chemical substances, including endothelin-1, prostaglandin F2-alfa, sodium nitroprusside,6 histamine,7 ouabain, serotonin,8,9 and ketanserin.10
Electric field stimulation is widely used in physiologic and pharmacologic research, for example, studies of the effects of calcitonin gene–related peptide in a rat cremaster muscle model11 and investigations of the contractility of the pregnant rat uterus.12,13 A number of reports have been published on investigations of the physiologic and pharmacologic behavior of different vessels by means of electric field stimulation, including the iridial arteries of the rat,14 the small mesenteric veins of the rabbit,15 the dog mesenteric artery and the rabbit ear artery,16,17 and the rabbit mesenteric artery and aorta.18 We have found no data on the application of electric field stimulation to study human placental blood vessels in the literature.
The aim of this work was to develop an electric field stimulation model suitable for investigations of the contractility of placental veins and arteries, and of the direct effects of different pharmacologic agents on the placental blood vessels. Most investigations of electric field stimulation are undertaken to stimulate sympathetic/adrenergic nerve terminals and to evoke local release of transmitters.19 A valid reason for determining the effects of electric field stimulation is that the placental circulation appears to be under very little or no functional sympathetic control20,21; for example, there is no or only a very poor response of the vessels of this circulation to exogenous noradrenaline.22 Another reason for using electric field stimulation is to provide a means of constricting these vessels in a receptor-independent fashion prior to addition of a dilatator/constrictor agonist.
MATERIALS AND METHODS
Experimentation on human placentas was approved by the Institutional Review Board (permission No. 909/ 1998 of the Ethics Committee of the University of Szeged). Placentas were obtained from the delivery room of the Department of Obstetrics and Gynecology, University of Szeged, immediately after birth; they were transferred in 500 mL icy Krebs-Henseleit buffer, and the experiments were begun 10–30 minutes after birth. All the placentas were selected randomly for study inclusion from term pregnancies of healthy, white mothers that ended with uncomplicated deliveries. The ages of the women from whom the placentas were obtained ranged between 19 and 28 years, with an average of 25.3 years. The gestational age at delivery was between 37 and 40 weeks, with an average of 38.1 weeks. The vast majority (80%) of the mothers were primiparas. The numbers of placentas included in each series of experiments are indicated by “n” in each figure caption. At least eight placentas were used for each series of experiments, and sometimes 12. We obtained four vessel rings from each placenta. After the umbilical cord had been cut off, thin polyethylene cannulae were led into the vein (with larger diameter) and the two arteries (with smaller diameters) in the stub, to separate the veins and arteries on the fetal surface of the placenta. The vessels were prepared for in vitro measurement according to the method outlined by Angus and Wright.23 Rings 1–1.3 mm in diameter24,25 were dissected from the identified veins and arteries just before their heading toward the stem villi. The precise length of the rings (4 mm) was achieved by use of a fixed, double-bladed scalpel. The loose connective tissue was carefully removed under a cool fiber optic light source and binocular dissection microscope (10×). The rings were taken distally from the site of introduction of the cannulae, the endothelium therefore remaining intact.
Rat mesenteric arteries were dissected from 200–220-g Sprague-Dawley rats through laparotomy after the animals were killed. Rat studies were allowed by permission No. I.-74-8/2002 of the Animal Ethics Committee of the University of Szeged.
The rings were mounted diametrically (as ring preparations) between two platinum electrodes in an organ bath containing 10 mL Krebs-Henseleit buffer (in mmol/L: 118 NaCl, 5 KCl, 2 CaCl2, 0.5 MgSO4, 1 KH2SO4, 25 NaHCO3, 10 glucose, pH = 7.4). The organ bath was maintained at 37C. Carbogen gas (95% O2 = 5% CO2) or gas simulating the in utero conditions (Messer, Szeged, Hungary) (for veins: pO2= 38 mm Hg, pCO2= 43 mm Hg; for arteries: pO2= 22 mm Hg, pCO2= 48 mm Hg)26 was bubbled through it. After mounting, the rings were equilibrated for 90 minutes before the experiment, and the buffer was changed every 10 minutes. The passive force was set at approximately 3.75g and 3.25g for veins and arteries, respectively. The optimal degree of stretch was ascertained by determining a contraction versus passive force curve in response to an electric field stimulation stimulus with a stimulating potential of 30 V, a period of 4 seconds, and a pulse width of 80 milliseconds. Using the same technique, the optimal passive force for rat mesenteric arteries turned out to be approximately 2.2g. These passive forces were similar to those used in previous electric field stimulation studies on other mammalian vascular smooth muscle preparations.27
Contractions were elicited by a digital, programmable stimulator (ST-02, Experimetria, London, United Kingdom). The force of the vessel rings was measured with a gauge transducer (SG-02, Experimetria U.K.), and recorded by an ISOSYS Data Acquisition System (Experimetria U.K.).
Measured or calculated points were plotted, and curves were fitted to these points with GraphPad Prism 2.01 software (San Diego, CA). Data were statistically analyzed with SPSS for Windows 9.0 (SPSS, Chicago, IL). Two- or three-way repeated measures analysis of variance was used to evaluate the significance levels of differences in all the figures except Figure 3. In Figure 3, two-way analysis of variance was used. Probability values lower than .05 were considered significant.
The optimal period time (interval between two stimuli) was determined by decreasing the applied period time until the individual contractions fused to produce a smooth contractile response. Twenty and 30 V were used as stimulating potentials (both supramaximal). The pulse width (the duration of a single stimulus) values used to elicit half-maximal contractions were determined by applying stimuli of different durations (pulse width: 25, 50, 100, and 200 milliseconds), and the elicited contraction responses were registered.
The effects of sodium nitrite (NaNO2) (3 × 10−7−10−3 mol/L) were studied. NaNO2 was administered in a cumulative way. The first dose was added at half-maximal contraction and the next doses every 5 minutes. All pharmacologic compounds were purchased from Sigma Aldrich, Budapest, Hungary. The length of the study enrollment period was 11.5 months (348 days).
Electric field stimulation induced fast and reproducible contractions in the human placental blood vessel rings. The optimal period time was found to be 5 seconds. For both tested stimulating potentials (20 V and 30 V), the strength of the contractions was plotted against the logarithm of the pulse width, and a sigmoidal curve was fitted. The contraction versus pulse width curve was analyzed, and the pulse width location at half-maximal force was calculated with GraphPad Prism 2.01 software. With stimulation at 20 V, the pulse width location at half-maximal force was greater than 500 milliseconds for both veins and arteries. These values were so extremely high that the curve was considered unsaturable (Figure 1). With stimulation at 30 V, the optimal pulse width (the pulse width location at half-maximum force) for veins and arteries was 100 milliseconds and 119 milliseconds, respectively (Figure 2). The contractions were not changed by tetrodotoxin (10−6 mol/L). Pre-treatment with verapamil (10−6 mol/L), which is a blocker of voltage-operated (L-type) calcium (Ca2+) channels or nickel (Ni2+) (2 mmol/L) (nonselective blocker of cation channels), inhibited the contractions to a magnitude of 63.81% ± 7.69% and 88.36% ± 12.17% (mean ± standard error of the mean), respectively. Combined verapamil and Ni2+ treatment inhibited the contractions to a similar magnitude as Ni2+ treatment by itself. In Ca2+-free medium after combined cyclopiazonic acid (10−5 mol/L) (depletes Ca2+ through inhibition of sarcoplasmic reticulum Ca2+-adenosinetriphos-phatase) and Ni2+ treatment, it was not possible to elicit contractions with electric field stimulation (Figure 3).
With stimulation at 20 V, the contractions of the veins were significantly stronger than those of the arteries at all applied pulse widths (P < .05). With stimulation at 30 V, the contractions of the veins were stronger at all applied pulse widths, but none of the differences were significant (P > .05).
After reaching half-maximal contraction, the placental vessel rings exhibited a time-dependent spontaneous relaxation, despite continuous stimulation. The arteries displayed a slightly greater relaxation, but the difference between the relaxation of the veins and the arteries was not significant (P > .05). The proportion of the spontaneous relaxation as a function of time after the half-maximal contraction was reached could be described by two (veins and arteries) third-degree polynomial functions (y = A + Bx + Cx2 + Dx3) (Figure 4) (second-degree polynomial functions are also appropriate). According to these functions, the proportion of the spontaneous relaxation could be determined at any moment within 40 minutes after the half-maximal contraction.
Bubbling through physiologic in utero hypoxic gases significantly enhanced the contractile responses of both the human placental vein and artery rings to electric field stimulation at all applied pulse widths (Figure 2), though the optimal pulse widths were not changed significantly (99 milliseconds and 105 milliseconds for veins and arteries, respectively). The spontaneous relaxation of the veins was not altered, but those of the arteries were reduced to zero (Figure 4).
Electric field stimulation (with the same stimulating parameters as used for placental vessel rings) also induced fast and reproducible contractions on the rat mesenteric arterial rings. After reaching half-maximal contraction, the rat mesenteric rings also exhibited a time-dependent spontaneous relaxation, despite continuous stimulation. The proportion of the spontaneous relaxation as a function of time after the half-maximal contraction was reached could be described by another third-degree polynomial function. Contrary to the placental vessel rings, in utero gases significantly blunted the forces of the contractions at all applied doses (Figure 5), but they did not alter the spontaneous relaxation of the rat mesenterial arterial rings (Figure 6).
The inhibitory or stimulatory effects caused by any drug itself, besides the spontaneous relaxations, could be calculated by correcting the measured contractions by the third-degree polynomial functions. The correction procedure was as follows: 1) multiplication of the actual contractions by the proportions of the spontaneous relaxation measured 5, 10, 15, 20, 25, 30, 35, and 40 minutes after addition of the first dose. The results of these multiplications were the actual spontaneous relaxations, which could have been measured without addition of the drugs; 2) subtraction of the actual spontaneous relaxation from the actual measured relaxation, leaving the actual relaxation caused by the drug itself; and 3) division of the actual drug-caused relaxation by the actual contraction, resulting in the proportions (percentages) of the drug-caused relaxation, which were plotted against the logarithms of the concentrations (semilogarithmic dose–response curve).
NaNO2 antagonized the contractions of the placental vessel rings in a significant and dose-dependent manner, but the efficacy of NaNO2 was significantly decreased by the in utero gases (Figure 7).
The electric field stimulation method has the advantage that another pharmacologic agent is not needed to elicit contractions, and interactions between the contractile agent and the examined pharmacons can therefore be avoided. In this study, we examined the effects of electric field stimulation on human placental blood vessels obtained from uncomplicated term pregnancies. Small-diameter resistance vessels, compared with conduit vessels, probably contribute more to the hemodynamics of placental bed perfusion, though the question of the location and the diameter of resistance vessels is, in general, unsolved, and the feed arteries can be as active in flow control as the microvasculature.28 According to recent data, the vessel rings obtained by us can be considered both as conduit and resistance vessels. In addition, evidence that larger or smaller segments contribute to a lesser or greater degree to changes in the blood flow of human organs (including the placenta) is not available.28 The optimal parameters of electric field stimulation were then determined. It has been proven that in oxygenated Krebs-Henseleit buffer, electric field stimulation does not generate substances that change the contractile state of the smooth muscle.29 To cause less harm to the tissue sample, mild stimulation was desirable. The lower the frequency (the longer the period time) of the stimulation, the less stressful it is to the tissue.
To investigate both the dilatator and the constrictor effects of the pharmacons, half-maximal contractions were needed. The pulse width location at half-maximal force of these contraction versus pulse width curves were considered to be the optimal pulse widths. With stimulation at 20 V, the contraction versus pulse width curve proved to be unsaturable, and it was therefore impossible to determine the optimal pulse widths. With stimulation at 30 V, the contraction versus pulse width curve was saturable; accordingly, 30 V was chosen as the optimal stimulating potential in our further experiments, despite the fact that the higher stimulating potential might be more harmful to the tissue. The assessed optimal pulse width parameters conform to the theory that the use of a short pulse width (0.7–5 milliseconds) is thought to selectively stimulate nerves but not smooth muscle, which requires a much longer pulse width (60–133 milliseconds) for direct excitation.30 Though with the high pulse widths and voltages applied, responses to electric field stimulation were not likely due to stimulation of nerves, the non-neurogenic nature of the contractions was checked functionally by blocking the nerve action potentials conduction with tetrodotoxin (10−6 mol/L). These results therefore support previous studies, which suggested an absolute lack of sympathetic innervation in the placental circulation.20,21 The involvement of extracellular and/or intracellular Ca2+ and a possible subsequent opening of voltage-operated Ca2+ channels was assessed by conducting experiments in Ca2+-free medium and with blockers of both voltage-operated Ca2+ channels and the intracellular Ca2+ stores. The results of these experiments suggested that the direct, non-neurogenic contractile effect of electric field stimulation on isolated human placental blood vessel rings mainly depends on the influx of extracellular Ca2+ via voltage-operated Ca2+ channels, partly on the mobilization of intracellular Ca2+ stores, and on a mechanism independent of intracellular Ca2+ concentration elevation. Because of the approximately 25% difference between the inhibitory effects of verapamil and Ni2+, this mechanism independent of intracellular Ca2+concentration elevation is likely to be the influx of other extracellular cations besides Ca2+. In our experiments, veins gave stronger contractile responses than arteries. These functional results confirmed the morphological properties of the walls of the placental veins and arteries. Force-producing smooth muscle cells lie within the media,31 and the smooth muscle–containing tunica media is thicker in placental veins than in arteries.32 The clinical relevance of the differences between the contractions of the veins and the arteries in the testing of future drugs is difficult to predict, because the response of the vessels to drug actions depends not only on the muscle layer thickness but also on the densities of different receptors, the activities of second messenger systems, etc. These parameters differ for every pharmacon. Otherwise, in general it might be stated that drugs that can alter the diameter either of the veins or the arteries may change the placental blood flow. Increased blood flow may be beneficial in preeclampsia and intrauterine growth restriction.
The parameters needed for half-maximal contractions of the veins and arteries were different, but the difference was not significant. The spontaneous relaxation after the half-maximal contractions were reached did not display a significant difference either.
Because the placenta is a hypoxic organ, the effects of in utero gases on human placental blood vessel rings were examined. In addition, as a comparison, the effects of the same gases were also investigated on rat mesenteric arterial rings. Interestingly, the results of these experiments suggested that, contrary to other, nonplacental vessels, in utero physiologic hypoxic circumstances have a stimulatory/enhancing effect on the contractility of human placental vessels. These results also suggest that during electric field stimulation studies on human placental blood vessels in utero, gases should be used instead of carbogen gas.
To test the pharmacologic capability of our model, the effects of a nitrovasodilator (NaNO2) was examined.
NaNO2 is a well-known nitric oxide donor. As might be expected, it antagonized the electric field stimulation contractions of the placental vessel rings in a significant and dose-dependent way. The efficacy of NaNO2 was significantly decreased by the in utero gases, leading us to the conclusion that the contractions of human placental blood vessel rings are better under physiologically hypoxic in utero conditions. The relaxant effect of NaNO2 was more pronounced on placental veins than on arteries at all applied doses, but none of the differences reached the level of significance. This was in agreement with data from the literature, that placental vessels obtained from normotensive pregnancies are sensitive to the relaxant effect of nitrovasodilatators (glyceryl trinitrate, sodium nitroprusside, and S-nitroso-N-acetylpenicillamine).33 These results also confirm the benefit of using in utero gases during human placental blood vessel electric field stimulation studies.
From our present findings, it may be concluded that we have successfully applied electric field stimulation for the study of human placental vessels, which is therefore a new experimental possibility for investigations of the direct placental vascular effects of different pharmacologic agents.
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