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. Author manuscript; available in PMC: 2013 May 22.
Published in final edited form as: EJ Neonatol Res. 2012 Jan;2(1):13–24.

Spontaneous Rhythmic Contractions (Vasomotion) of the Isolated, Pressurized Ductus Arteriosus of Preterm, but Not Term, Fetal Mice

Megan Vucovich 1, Noah Ehinger 2, Stanley D Poole 2, Fred S Lamb 2, Jeff Reese 2,3
PMCID: PMC3661283  NIHMSID: NIHMS443948  PMID: 23710420

Abstract

The mechanisms that regulate relaxation of the fetal ductus arteriosus (DA) and its postnatal constriction are the subject of ongoing studies. Using pressure myography, a pattern of rhythmic oscillatory contractions termed vasomotion was observed in the isolated DA of preterm (day 15) fetal mice. Vasomotion was enhanced by oxygen-induced DA constriction and other contractile agents, and diminished by vasodilatory stimuli or inhibition of chloride channels. The DA of late preterm (day 17) or term (day 19) gestation fetal mice did not exhibit vasomotion. These studies establish the stage-specific presence of vasomotion in the DA of fetal mice and suggest that complex events contribute to intrinsic mechanisms for control of fetal DA tone.

Keywords: ductus arteriosus, preterm, mouse, vasomotion, nitric oxide, prostaglandin, chloride channel, niflumic acid

Introduction

The ductus arteriosus (DA) is a fetal vascular conduit that interconnects the pulmonary artery and the descending aorta, allowing oxygenated blood from the placenta to bypass the high resistance pulmonary vascular bed in utero to provide the peripheral circulation with a fresh supply of oxygen and nutrients. Compared to the elastic great arteries of the outflow tract, the DA is a large muscularized artery with multiple layers of spirally-arranged smooth muscle cells. Ductus smooth muscle cells constrict after birth to achieve permanent closure of the lumen, sealing off the shunt between the aortic and pulmonary circulations. Although the phenomenon of postnatal DA closure has been studied for centuries,1,2 the mechanisms that regulate DA smooth muscle tone have only begun to be revealed over the past few decades.3-5

Maintenance of DA patency in utero is essential for fetal well-being. Blood vessel caliber is the product of intrinsic and extrinsic regulators of vascular tone; these factors are the subject of many ongoing studies. Intrinsic mechanisms for the control of blood flow include metabolic, myogenic, and endothelial contributions to muscular tension in the vessel wall. Studies on the ex vivo DA in our laboratory and others6-8 demonstrated the presence of pressure-induced tone, or the myogenic response, which maintains an optimal DA lumen diameter in the presence of alterations in distending pressure. The myogenic response is an important autoregulatory mechanism in muscular arteries of different vascular beds. Our observations were made using cannulated, pressurized vessel myography.6

Recently, our studies on the isolated DA of fetal mice also revealed the presence of sustained, spontaneous rhythmical contractions. Repetitive cycles of brisk contraction and relaxation occurred once the vessels were pressurized and equilibrated, but were not present in all preparations. These findings are consistent with the vascular process known as vasomotion – an oscillatory change in vascular tone that has been reported in many vascular beds. These synchronized changes in blood vessel tone are reported to have positive effects on oxygen delivery and may be protective during ischemia.9-11 Altered vasomotion has been observed in diabetes, hypertension, obesity, acidosis, and other pathological states, using both in vitro and in vivo assessments.12-14 To the best of our knowledge, vasomotion has not been described in the DA of any species. In this report, we describe the presence of vasomotion in the murine DA and characterize the features of this process in preterm fetal mice.

Materials and Methods

Animals and tissues

Experiments were conducted in accordance with National Institutes of Health animal care standards and were approved by the Institutional Animal Care and Use Committee at Vanderbilt University Medical Center. Adult female CD1 mice (age 48– 60days; Charles River, Raleigh, NC) were bred with fertile males to produce timed pregnancies (copulatory plug = day 1 of pregnancy). Pregnant females were anesthetized by i.p. injection of 0.4 ml of 2.5% avertin (2,2,2-tribromoethanol in tertamyl alcohol; Sigma-Aldrich, St. Louis, MO), followed by isoflurane inhalation (Baxter, Deerfield, IL) to promote fetal anesthesia. Dams were killed by cervical dislocation at 0900h on days 15, 17, or 19 (“term”) of pregnancy. Fetuses were delivered by uterine incision. Any fetus that displayed spontaneous respiratory efforts after delivery or had signs of lung inflation at the time of dissection were excluded from further study.

For fresh ex vivo myography studies, fetuses were removed from the uterus and immediately submerged in ice-cold, deoxygenated (95% N2, 5% CO2) Krebs buffer and secured in supine position in a dissection dish. Krebs buffer was modified (in mM: 109 NaCl, 34 NaHCO3, 4.7 KCl, 0.9 MgSO4, 1.0 KH2PO4, 11.1 dextrose, and 2.5 CaCl2) to maintain a stable pH (7.25-7.30) in the presence of a closely monitored bubbling rate required to maintain relative hypoxia in the vessel bath (dissolved oxygen content = 1.5-1.8%; measured PO2 = 38-45 Torr). The remaining pups were stored in chilled buffer for later dissection. In these mice, a transverse abdominal incision was made and the diaphragm was opened, so as to ensure unobstructed flow of buffer into the thorax until dissection took place. Under stereomicroscopy, transverse abdominal and lateral chest wall incisions were made to expose the thoracic contents. The DA and branch pulmonary vessels were separated from surrounding tissues. Isolation of the fetal ductus was performed by first dissecting a small region of cardiac tissue inferior to the pulmonary valve. The aorta was cut on either side of the junction with the DA. The isolated main pulmonary artery-DA-transverse aorta segment of vascular tissue was freed from any remaining tissues. At all times, care was taken to avoid excess tension or stretch of vascular tissues during dissection.

Vessel studies

Isolated vascular segments were transferred to custom-made microvessel perfusion chambers (Instrumentation and Model Facility, University of Vermont) that were flushed with chilled, deoxygenated Krebs buffer (4 ml capacity). Glass micropipettes (1.2 mm, World Precision Instruments, Sarasota, FL) were pulled to 120 or 170 mm diameter for preterm and term vessels, respectively. Myocardium at the base of the pulmonary artery was used to handle the excised tissues and position the vessel preparation on the proximal pipette. Care was taken to prevent movement of the pipette tip beyond the branch pulmonary arteries into the DA lumen. Tissues were secured in position using a single strand taken from 000 braided nylon suture. Micromanipulators were used to position the aortic end of the vessel preparation near the distal cannula tip. The orifice of the descending portion of the transverse aorta was then advanced onto the distal pipette and secured. The branch pulmonary arteries were tied back to the proximal pipette, then the DA was secured at its junction with the transverse aorta, resulting in isolation of the DA between the pipette tips.

Once mounted, vessels and perfusion chambers were transferred to an inverted trinocular microscope equipped with an analog video camera and computer-assisted image capture system (IonWizard 6.2, IonOptix, Milton, MA) to continuously record the intra-luminal diameter of the DA. Optical markers used to detect the lumen diameter were positioned at the endothelial-smooth muscle interface during full vessel closure, such that -85 to -90 % change from baseline reflected complete constriction of the ductus. Vessels were pressurized by a column of deoxygenated Krebs buffer, with continuous recording of distending pressure (in mmHg). Bath temperature was held constant at 36.5-37.5 C and was continuously monitored by a thermal microprobe placed immediately adjacent to the mounted vessel. Chambers were perfused with warmed deoxygenated Krebs buffer at 6 ml/min. via low gas permeability Tygon tubing. The head gas from the bubbling reservoir was routed to flow over the perfusion chamber to maintain constant oxygen conditions. Oxygenation of the bath solution was determined by dissolved oxygen content using a 2 mm electrode (inO2; Innovative Instruments, Tampa, FL) and by periodic blood gas determination (Radiometer ABL700; Westlake, OH). Term gestation vessels were pressurized to 5 mmHg and allowed to equilibrate at 37 C for 30-40 minutes in constant flow, non-recirculating, deoxygenated Krebs buffer. Preterm vessels were equilibrated at 2 mmHg. A stepwise increase in distending pressure was applied in 2 mmHg (preterm vessels) or 5 mmHg (term vessels) increments up to the appropriate working pressure (6 mmHg, preterm; 20 mmHg, term) with 10-15 minutes of equilibration at each pressure. Vessels were exposed to 50 mM KCl in deoxygenated Krebs for 3-5 minutes (with 1-2 repetitions) and the contractile response for each vessel preparation was determined. After return to baseline, the bath was changed to a recirculating buffer circuit (total volume 20 ml) for treatment with various drugs.

Drugs and compounds

Krebs buffer was adjusted to maintain isotonicity with increased KCl solutions. KCl-, oxygen-, or drug-induced vessel constriction was maintained until a stable baseline vessel diameter was established. Vessel studies were performed with established doses for the non-specific NOS inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME) (10-4 M; Cayman Chemical, Ann Arbor, MI), the nonspecific COX inhibitor, indomethacin (10-5 M; Sigma-Aldrich, St. Louis, MO), the synthetic thromboxane receptor agonist, U-46619 (10-7 M; Cayman), niflumic acid (10-4 M; Sigma-Aldrich) or papaverine (10-4 M; Sigma-Aldrich). Indomethacin and U46619 were dissolved in ethanol; all other drugs were dissolved in water. Final solvent concentration in the bath was limited to 0.01% for each drug; the maximum cumulative concentration of solvent for any repetitive drug study was 0.02%. Vessels were continuously exposed to each agent until a stable baseline was reached (typically 20-30 minutes). A final KCl stimulation was given to confirm vessel responsiveness at the end of the study, followed by exposure to papaverine, to determine maximum vessel diameter. Vessels that did not respond to the final KCl stimulus within 10% of the initial percent change from resting baseline were excluded from further consideration.

Results

The pressurized term and preterm DA exhibit intrinsic control of vasomotor tone

The isolated fetal DA at term gestation demonstrated the presence of pressure-induced tone (myogenic response) with each stepwise increase in distending pressure. (Fig. 1a) Preterm DAs displayed a similar response. Following equilibration, exposure to 50 mM KCl induced rapid, strong constriction of the term DA. Complete lumen closure was frequently observed when the measured intraluminal diameter was less than 50 mm. The preterm DA also had a brisk response to KCl but the lumen was not fully closed. (Fig. 1b) Thickening of the muscular wall was evident during closure of the term DA (Movie 1), and was noted to a lesser extent in the preterm DA. (Movie 2) Wall thickening and vessel constriction were fully reversible when the depolarizing KCl solution was washed off and replaced by recirculating Krebs buffer. At the end of each experiment, stimulation with 50 mM KCl confirmed vessel responsiveness, followed by complete vessel relaxation in response to the direct-acting smooth muscle relaxant, papaverine6 (not shown), indicating that the pressurized term and preterm DA appear to actively maintain an optimal tone and lumen diameter under in vitro experimental conditions.6

Figure 1.

Figure 1

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Oxygen-induced constriction reveals vasomotion in the preterm DA. Representative tracings of isolated vessels that were equilibrated, pressurized, stimulated with 50mM KC1, and then exposed to increasing oxygen concentrations in the myography bath. A myogenic contractile response was noted in both term and preterm DAs during the ramp-up to working pressure. Exposure to increased oxygen resulted in progressive DA constriction. Oxygen exposure was associated with rhythmic contractions in the preterm but not the term DA. Vasomotion abated during wash-off to baseline myography conditions.

Movie 1. Closure of the term gestation (d19) fetal DA.

Movie 1

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The isolated term DA was mounted, equilibrated to bath conditions and pressurized. Membrane depolarization in response to 50mM KCl prompted brisk constriction of the DA, resulting in increased wall thickness and occulusion of the lumen. Complete lumen closure was reversible upon wash-of to regular Kreb's buffer. [Editor' Note: movies can only be viewed in the HTML version of this manuscript, please go to http://neonatologyresearch.com/?page_id=2278 to view this movie.]

Movie 2. Constriction of the preterm (d15) fetal DA.

Movie 2

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The isolated preterm DA was mounted, equilibrated to bath conditions and pressurized. Membrane depolarization in response to 50mM KCl prompted rapid constriction of the DA, resulting in wall thickening and narrowing of the lumen. Lumen constriction was reversible upon the wash-off to regular Kreb's buffer. [Editor's Note: movies can only be viewed in the HTML version of this manuscript, please go to http://www.neonatologyresearch.com/?page_id=2278 to view this movie.]

Vasomotion is present in the preterm DA, but not the term DA

The isolated term DA achieved a stable baseline diameter after equilibration, stepwise increase to working pressure, and exposure to KCl. Minimal fluctuation was noted in the lumen diameter under steady-state conditions and there were no repetitive contractions observed in any preparation (n=8). In contrast to full-term vessels, preterm (d15) vessels underwent sustained cyclic episodes of vascular constriction and relaxation at baseline (11 out of 14 preparations), so that at rest the vessel appeared to be twitching (Fig. 1b) The amplitude of the rhythmic contractions was relatively constant once stable conditions were achieved. The frequency of contractions was variable (range 3-7 per 10 min epoch). The isolated DA of fetuses with less severe prematurity (d17 of gestation) did not demonstate vasomotion under any conditions (n=7; data not shown). Vasomotion was not observed until preterm (d15) vessels underwent a prolonged period (∼2 hr) of equilibration, pressurization, stimulation with KCl, and exposure to an agonist. Additionally, it was noted that DAs obtained from fetuses that were set aside for later dissection frequently did not display vasomotion, despite being responsive to KCl, oxygen, U46619 and other stimulants, in contrast to the freshly mounted vessels, which consistently exhibited vasomotion.

Preterm vasomotion characteristics are altered by contractile or vasodilatory stimuli

Exposure to increasing oxygen content in the perfusion bath caused sequential reduction in the lumen diameter of both term and preterm Das. (Fig. 1) In vessels from preterm (d15) fetuses, oxygen-induced DA constriction was accompanied by an increase in the frequency of rhythmic contractions. (Fig.1 b) The amplitude of each contraction was diminished as overall DA dimensions were reduced in response to increased oxygen exposure. At the completion of each experiment, a wash-off step and return to baseline conditions (Krebs buffer aerated with 0% oxygen) caused cessation or reduction in vasomotion. (Fig. 1b)

Preincubation of isolated term and preterm DAs with the thromboxane receptor mimetic, U46619 (10−8 M), produced approximately 60% of maximal contraction. (Fig. 2) U46619-induced constriction of the term DA did not produce vasomotion (Fig.2a), similar to term DAs that were constricted by oxygen exposure (Fig.1a). However, preterm (d15) DAs developed spontaneous rhythmic contractions upon exposure to U46619. (Fig. 2b) The frequency and amplitude of O2- and U46619-induced contractions were similar. (Fig. 1b and Fig 2b, respectively)

Figure 2.

Figure 2

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Altered vasomotion in response to PGE2. Representative tracings of isolated DAs that were equilibrated, pressurized, and treated with the thromboxane receptor agonist, U46619 (10−8 M), to induce submaximal constriction (arrowhead). Preconstricted vessels were then exposed to increasing concentrations of PGE2 (10−12 to 10−6 M; arrows). Vasomotion in the isolated preterm DA decreased and then disappeared with advancing PGE2 doses.

Term and preterm DAs that were submaximally pre-constricted by U46619 underwent concentration-dependent relaxation in response to increasing doses of PGE2 (10−12 to 10−6 M). Progressive exposure to PGE2 eventually produced dilation of the isolated DA beyond its baseline diameter. (Fig. 2) However, in preterm (d15) DAs, the initial response to PGE2 was reduction in the frequency of vasomotion. (Fig. 2b) A change in vasomotion frequency was typically observed at a range of 10−11 to 10−10 M concentrations of PGE2. At 10−8 to 10−7 M PGE2, vasomotion ceased and DA dilation was observed. The behavior of preterm (d15) DAs that were pre-constricted with U46619 (n=5) (Fig. 2b) was similar to DAs that were pre-constricted with 12% oxygen (n=6) (O2 pre-constriction not shown).

Vasomotion is dampened by chloride channel inhibition, but not by inhibitors of prostaglandin or NO synthesis

Once vasomotion was established, treatment with the non-selective cyclooxygenase (COX) inhibitor indomethacin, or the non-specific inhibitor of NOS isoforms, L-NAME, did not have an effect on the rhythmic contractions of preterm (d15) DAs (n=3; not shown). In contrast, exposure to niflumic acid, which has NSAID properties but also inhibits chloride channel currents and at higher concentrations can activate potassium channels,15 caused reduction in the frequency of vasomotion (3 × 10−5 M). (Fig. 3 and Movie 3) Exposure to a higher dose of niflumic acid stimulated mild dilation of the DA (10−4 M), in contrast to indomethacin, which causes the expected NSAID-induced constriction of the isolated fetal DA at these doses.6

Figure 3.

Figure 3

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Altered vasomotion in response to niflumic acid. Representative tracing of an isolated preterm (dl5) DA that was equilibrated, pressurized, and stimulated with 50mM KCl. Spontaneous vasomotion typically developed after warm-up and accommodation to recirculating bath conditions. Vessels were then exposed to standard doses of niflumic acid (NFA), an inhibitor of chloride channel current (10−5 to 10−4 M). Vasomotion in the isolated preterm DA diminished in response to 30 mM NFA and disappeared at 100 mM NFA. Brief vessel spasm was noted after wash-off to baseline conditions.

Movie 3A & B. Effect of Niflumic Acid.

Movie 3A & B

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Spontaneous vasomotion typically developed after warm-up and accommodation to recirculating bath conditions.(A) This oscillating DA was then exposed to niflumic acid (NFA), an inhibitor of chloride channel current (10−5 to 10−4 M). Vasomotion diminished in response to 30 mM NFA (second dose) and disappeared at 100mM NFA (third dose). (B) Brief vessel spasm was noted after wash-off to baseline conditions (small red hash marks along x-axis denote drug administration or wash-off). Exposure to 50mMKCI at the end of the experiment demonstrates viability of the ex vivo preparation. [Editor's Note: movies can only be viewed in the HTML version of this manuscript, please go to http://www.neonatologyresearch.com/?page_id=2278 to view this movie.]

Discussion

The main findings of this paper are that vasomotion occurs in the isolated DA of early preterm (d15) fetal mice, but not in the DA of late preterm (d17) or term (d19) gestation fetal mice. Video recordings highlight the rhythmical brief contractions that exemplify this process. As is typical of vasomotion, the contractile phase of the response was more rapid than the relaxation phase. Drugs or conditions that increased vascular tone contributed to DA vasomotion, while a wash-out to baseline conditions in the myography bath or exposure to increasing doses of PGE2 quelled DA vasomotion. The reduction in vasomotion induced by PGE2 occurred at lower concentrations than those required to cause vasodilation. Similarly, the inhibitory effect of niflumic acid on vasomotion occurred at a concentration (30 mM) that is relatively selective for blockade of chloride over potassium channels indicating a potential role for chloride channels in DA tone. The physiological significance of vasomotion in the preterm DA is unclear. However, these experiments provide new information on a process that is important in other vascular beds, and may form the basis for a better understanding of the mechanisms that regulate fetal DA tone or development.

It was originally thought that the fetal DA was a passive vessel that was maintained in an open position by hemodynamic forces.3,16 Later studies on ex vivo preparations of the fetal DA from different animal species identified various drugs and compounds that stimulated contraction of strips or rings of fetal DA tissues,17-20 suggesting that relaxation of the fetal DA was actively maintained by biochemical stimuli that oppose muscular constriction. Vasomotor tone of the fetal DA was also considered to have a neural component, based on the response to various neurotransmitters and studies that identified anatomical patterns of innervation.21,22 The discovery that prostaglandins stimulate relaxation of the DA23 and that inhibition of the prostaglandin synthase (COX) enzymes by non-steroidal anti-inflammatory drugs (NSAIDs) cause DA constriction24-26 solidified a growing body of evidence that active forces maintain relaxation of the fetal DA.4,5 Relaxation of the fetal DA is primarily maintained by nitric oxide, vasodilatory prostaglandins, the activity of certain ion channels, and low oxygen tension, but is also affected by adenosine, carbon monoxide, catecholamines, and generalized vessel immaturity.5 Despite the critical importance of DA tone in utero, we are not aware of any information on the presence of vasomotion in the fetal DA.

The muscular nature of the DA and the balance between vasodilatory and contractile forces that underlie baseline DA tone results in a vessel that is strongly susceptible to contractile influences. By comparison, the similar-sized fetal aorta has limited responses to contractile stimuli, whereas the fetal DA can undergo such severe constriction that the lumen is completely obliterated. The developmental stage at which the DA gains this degree of “muscular maturity” is unclear. In humans, the fetal DA is not particularly susceptible to NSAID-induced DA constriction until after 32-34 weeks of gestation.27,28 The same is true in fetal mice, where prostaglandin inhibition induces profound DA constriction in utero on d18 and d19 of gestation, but not at earlier stages.29 The opposite relationship exists for NO, such that inhibition of nitric oxide synthase causes moderate DA constriction at mid-gestation (d15) but minimal effects at term.6,30,31 In the present studies, we only observed vasomotion in the early preterm (d15) DA. Inhibition of NO and prostaglandin synthesis did not have an effect on the frequency or amplitude of vasomotion, suggesting another mechanism for rhythmic contractility in the DA wall.

Vasomotion is an oscillation in vascular tone that is driven by intrinsic forces in the vascular wall, and occurs independently from heartbeat, respiratory rate, and neural input. The rhythmic pattern of vasomotion may be regular or chaotic.32 Vasomotion has been recognized for over a century, and is reported in the microvasculature of many tissues from various different species. Large muscular arteries, including coronary, mesenteric, carotid, and pulmonary arteries also display vasomotion.12,33 A common mechanism has not been identified to explain the vasomotion process in diverse vascular beds. However, recent reviews highlight a number of mechanisms that have been invoked to explain these oscillatory contractions, including coordinated waves of intracellular calcium release and synchronization of adjacent smooth muscle cell contractions via gap junction proteins.12-14 Depending on the tissue, increased intracellular calcium ([Ca2+]i) levels may be accomplished by Ca2+ release from the sarcoplasmic reticulum (a cytosolic oscillator), along with Ca2+ influx through voltage-dependent Ca2+ channels (a membrane oscillator). Calcium-activated chloride channels stimulate smooth muscle contraction34,35 and can also serve as membrane oscillators that may act as underlying mediators of vasomotion.36-39 Our preliminary results suggest that inhibition of Ca2+-activated chloride current by niflumic acid slows (30 mM) and then stops (100 mM) vasomotion in the preterm fetal DA. The higher concentration of niflumic acid was associated with overt dilation of the DA raising the possibility that the drug also activated potassium channels.15 Due to the lack of specific and selective anion channel blockers, additional studies using other Cl− channel and transport inhibitors, siRNA approaches, or Cl− anion substitution buffers will be required to definitively demonstrate the role of Cl− in DA vasomotion.

The physiological significance of vasomotion is not fully understood. Studies performed in humans and animal models reveal an important relationship between high blood pressure and the propensity for blood vessel oscillation.40-46 Diabetes in both humans and animal models is associated with diminished vasomotion.13,14,47,48 Abnormal patterns of vasomotion have also been observed in patients with sympathetic dysfunction, Raynaud's syndrome, and in states of metabolic acidosis or poor tissue perfusion.12-14,49 Vasomotion may be beneficial for perfusion and oxygenation of peripheral tissues 10,12,50,51 or tissue fluid homeostasis.52,53 Modeling studies also suggest that oscillating vessels can facilitate flow with less resistance,54 yet a role for vasomotion in the DA is not clear. The fetal DA is a muscular, contractile vessel. Drugs that cause constriction of the DA in utero force blood flow into the normally restrictive pulmonary vascular bed and results in fetal heart failure, adverse pulmonary vascular remodeling, and potential fatality. Constriction of the fetal DA is usually related to maternal NSAID ingestion,55,56 although spontaneous DA closure is also reported.57-59 Fetal DA constriction is usually reversible.60,61 Intermittent constriction and later reopening has been referred to as “winking” of the DA,62 but these events are unrelated to oscillatory contractions. Repetitive, pulsatile contractions of the isolated fetal mouse DA have been reported in response to oxygen exposure.63,64 However, these slow contractile waves are more reminiscent of spontaneous phasic contractions in coronary arteries.65-67

It is possible that vasomotion is an insignificant consequence of DA development, or that immature DA smooth muscle cells have incomplete mechanisms for coordinated regulation of fetal DA relaxation. On the other hand, the observation that vasomotion was limited to the early preterm DA may provide new insights into why the DA of extremely preterm infants responds poorly to treatment. This is a descriptive study that requires confirmation using larger numbers of vessels and more sophisticated approaches. However, our findings set the stage for additional investigation on the role of chloride channels in the fetal DA and the role that vasomotion plays in fetal DA tone or development.

Acknowledgments

This work was supported by NIH grants HL77395 and HL 96967 (JR)

Footnotes

Author Disclosure: The authors have nothing to disclose.

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