After birth, constriction of the full-term ductus arteriosus induces oxygen, glucose and ATP depletion, cell death, and anatomic remodeling of the ductus wall. The immature ductus frequently fails to develop the same degree of constriction or anatomic remodeling after birth. In addition, the immature ductus loses its ability to respond to vasoconstrictive agents, like oxygen or indomethacin, with increasing postnatal age. We examined the effects of premature delivery and postnatal constriction on the immature baboon ductus arteriosus. By 6 days after birth, surrogate markers of hypoxia (HIF1α/VEGF mRNA) and cell death [dUTP nick-end labeling (TUNEL)-staining] increased, while glucose and ATP concentrations (bioluminescence imaging) decreased in the immature ductus. TUNEL-staining was significantly related to the degree of glucose and ATP depletion. Glucose and ATP depletion were directly related to the degree of ductus constriction; while TUNEL-staining was logarithmically related to the degree of ductus constriction. Extensive cell death (>15% TUNEL-positive cells) occurred only when there was no Doppler flow through the ductus lumen. In contrast, HIF1α/VEGF expression and ATP concentrations were significantly altered even when the immature ductus remained open after birth. Decreased ATP concentrations produced decreased oxygen-induced contractile responses in the immature ductus. We hypothesize that ATP depletion in the persistently patent immature newborn ductus is insufficient to induce cell death and remodeling but sufficient to decrease its ability to constrict after birth. This may explain its decreasing contractile response to oxygen, indomethacin, and other contractile agents with increasing postnatal age.
after birth, the full-term ductus arteriosus closes in two consecutive phases, a constriction phase lasting several hours followed by an anatomic remodeling phase. The remodeling phase, which permanently closes the ductus, involves massive death of smooth muscle cells in the ductus muscle media and loss of ductus responsiveness to vasoactive stimuli (2, 3, 11, 18). In the late-gestation fetus, the ductus muscle media is supplied with oxygen and nutrients by diffusion from its lumen and vasa vasorum (9). Postnatal constriction of the full-term ductus obliterates its lumen, obstructs flow through its vasa vasorum, and increases wall thickness producing a marked increase in diffusion distances for oxygen and nutrients (11). Typically, the thickness of the avascular ductus wall increases from 0.48 mm (as a fetus) to 1.09 mm during the first hours after delivery (2, 11). This marked increase in diffusion distance produces severe oxygen, glucose, glycogen, and ATP depletion in the ductus muscle media (12). In vitro studies have shown that cell death and anatomic remodeling in the full-term ductus correlate best with ATP depletion; this is most marked when both tissue oxygen and glucose reserves are severely depleted (8, 12).
In contrast with the full-term ductus, constriction of the immature ductus frequently fails to cause luminal obliteration and only moderately increases diffusion distances for oxygen and nutrients within the muscle media (2, 11). The typical increase in avascular wall thickness (from 0.47 mm in the preterm fetus to 0.67 mm in the preterm newborn) is only one-third of that seen at term (2, 11). As a result, the postnatal diffusion distances in the preterm ductus are much less than they are at full term. This is associated with less cell death and less remodeling and leads to persistent ductus luminal blood flow after birth (1, 2, 14). In vitro studies suggests that preservation of ATP, glucose, and glycogen stores may explain why the immature ductus develops less cell death (8, 12). However, the actual degree of ATP, glucose, and glycogen depletion within the immature newborn ductus in vivo is currently not known.
In addition to the absence of anatomic remodeling, the immature newborn ductus progressively loses its ability to constrict, both in vitro (1) and in vivo (7, 15), when exposed to oxygen or indomethacin during the first days after birth. The explanation for the impaired contractility of the persistently patent immature newborn ductus is currently unknown.
In this study, we examined ductus from full-term and immature fetal and newborn baboons and lambs to determine how constriction of the ductus arteriosus affects its concentrations of oxygen, glucose, glycogen, and ATP in vivo; and, to determine how these changes relate to the appearance of cell death in the vessel’s wall and to the ability of the ductus to constrict after birth.
MATERIALS AND METHODS
All studies were reviewed and approved by the local Institutional Animal Care and Use committees on animal research at the University of California, San Francisco and the Southwest Foundation for Biomedical Research.
We used fetal and newborn baboons (Papio sp., full term = 185 day gestation) to investigate the effects of ductus constriction on hypoxia (HIF1α and VEGF mRNA expression), energy status (ATP, glucose, and glycogen depletion), and cell death [dUTP nick-end labeling (TUNEL) staining] (see TUNEL staining and enzyme-linked bioluminescence imaging). Animal care, surgery, and necropsy were performed as previously described (2, 16). Immature (125 ± 2 day gestation; 69% of full term) and mature (175 day gestation; 97% of full term) fetal baboons were delivered by cesarean section and euthanized before breathing. Mature full-term newborn baboons were euthanized between 1 and 2 days after spontaneous full-term delivery. Immature newborn baboons were delivered by cesarean section at 125-day gestation, intubated with a 2-mm endotracheal tube, and cared for in the primate intensive care nursery for the first 6 days after delivery. Intensive Care Nursery management was performed as previously described (2, 16). Ventilator management was designed to maintain the arterial blood gases in the following ranges: PaO2 = 50–80 Torr, PaCO2 = 35–55 Torr, and pH = 7.25–7.45. The immature newborn baboons were euthanized on day 6 after delivery. Immediately before necropsy, the preterm baboons still required mechanical ventilation with FiO2 = 0.38 ± 0.20, mean airway pressure = 9 ± 3 cmH2O, and rate = 33 ± 9 breaths/min.
At necropsy, the ductus was dissected in 4°C PBS solution and frozen in liquid nitrogen (for HIF1α and VEGF mRNA quantification) or embedded in Tissuetek (American Master Tech, Merced, CA) (for TUNEL staining and ATP, glucose, and glycogen analysis) and frozen in liquid nitrogen. The dissection procedure required ∼25 min before the tissue was frozen. The ATP, glucose, and glycogen levels within the ductus are likely to change during this period. However, the dissection protocol was identical for all animals so valid comparisons between groups could still be made.
Our primary goal was to examine the effects of ductus constriction on hypoxia, energy status, and cell death in the immature ductus by comparing immature fetal ductus with immature newborn ductus that either remained partially open after birth or that closed after birth. To assess ductus constriction, a complete echocardiographic exam, including assessment of ductal patency, was performed daily using an 8-mHz transducer interfaced with a Biosound AU3 (Genoa, Italy) echocardiographic system as previously described (20). The full-term newborn baboon ductus constricts rapidly after birth and is closed by Doppler exam within 12 h after delivery. In contrast to the full-term ductus, the immature ductus frequently remains open after birth. Therefore, on the basis of the Doppler exam, the ductus could be divided into five groups: mature fetus, mature newborn-closed, immature fetus, immature newborn-open, and immature newborn-closed. All of the immature ductus that were open at necropsy (on day 6) had been open on the five preceding daily Doppler exams.
We used the expression of HIF1α and VEGF mRNA as surrogate markers for the degree of tissue hypoxia since their expression correlates with the degree of hypoxia in the ductus and other tissues (17, 20). Total RNA was isolated from the frozen ductus of immature fetuses (n = 8), immature newborns whose ductus closed prior to necropsy (n = 6), immature newborns with a persistently open ductus (n = 10), mature fetuses (n = 8) and full-term 1- to 2-day-old newborns (n = 8), as previously described (20). TaqMan probes were designed using the Primer Express program (20). An ABI PRISM 7700 Sequence detection system was used to determine the number of PCR cycles required for product detection [cycle threshold (CT value)]. Reactions were carried out in triplicate. The fewer the number of starting copies of a gene’s mRNA, the higher the CT value required for product detection. All reactions were repeated on at least three separate days. Data were analyzed using the Sequence Detector version 1.6.3 program.
We used the baboon housekeeping gene MDH as an internal control to normalize the degree of HIF1α and VEGF expression since its CT value is constant throughout gestation and after birth (20). We used the method of relative gene expression, where ΔCT (MDH-gene) represents the difference in CT between MDH and the gene of interest (HIF1α or VEGF). Each unit of ΔCT (MDH-gene) represents a twofold increase in mRNA.
TUNEL staining and enzyme-linked bioluminescence imaging.
We determined cell death and energy status in the following frozen, embedded ductus: mature fetus (n = 3), mature newborn-closed (n = 2), immature fetus (n = 6), immature newborn-open (n = 6), and immature newborn-closed (n = 4).
We used the TUNEL technique (Apoptag Peroxidase detection system; Intergen, Purchase, NY) to detect cells in the early stages of DNA fragmentation and cell death, as we have described previously (12). We determined the number of TUNEL-positive nuclei, as a percent of muscle media nuclei, by examining more than 500 nuclei in the middle two quartiles of the ductus wall. Histologic measurements were made as close as possible to the point of maximal ductus narrowing determined by measuring the luminal area from serial (6 μm) cross sections of the frozen ductus (see below).
We used bioluminescence imaging to visualize and quantify the distribution of metabolites within the ductus wall. This method has been described in detail elsewhere (12). A brief methodological description is given below. Cryosections (15 μm) were mounted on poly-l-lysine slides, immediately fixed on a heating plate (95°C) for 10 min, and then stored at −20°C until analysis. To calibrate the bioluminescence signal, standards were made by dissolving different concentrations of ATP, glucose, or glycogen in PBS with 8% low molecular weight gelatin. The solution was frozen and 15-μm cryosections were made and treated in the exact same manner as the tissue sections.
To link the substrates of interest to the production of photons, different enzyme solutions containing luciferase were used. At the time of analysis, the desired enzyme solution was applied to the cryosection. The emitted photons were registered by a photon counting camera (model C2400–47; Hamamatsu Photonics, Japan) mounted on the microscope (Axiovert 135M; Carl Zeiss, Germany). The light intensity in different parts of the digital image reflects the local concentration of the studied metabolite (ATP, glucose, or glycogen). Standard curves were prepared from the mean bioluminescence intensity of the standard preparations. A darkfield image of the same section was also obtained to outline histological structures in the corresponding bioluminescence image. From every ductus, four consecutive sections were analyzed for each of the three metabolites. Each section was used for the analysis of one metabolite, and all measurements were performed at room temperature (23 ± 1°C).
In the ductus arteriosus, the point of maximal narrowing (smallest luminal area) occurs over a very short distance in the middle of the constricted vessel. Since multiple sections were needed to make all of the metabolic and cell death measurements on a single ductus, not all of the sections could be obtained from the region of maximal narrowing. Therefore, we determined the luminal areas of each of the frozen sections of ductus used for ATP, glucose, glycogen, and TUNEL analysis. This made it possible to compare measurements of ductus constriction at the same level as the analyses.
Sheep ductus rings: in vitro.
To examine how changes in ATP concentrations in the immature ductus might affect its ability to contract, we performed measurements of isometric tension in immature sheep ductus [at the same point in gestation (69% of full term) as the immature baboon ductus studied above] using an organ culture-isometric tension measuring system. We utilized the immature sheep ductus as a surrogate for the baboon ductus since the conditions for its incubation, stretch, contractility measurements, and oxygen and glucose concentrations had been previously established in our laboratory (8, 10). Mixed Western breed lamb fetuses (104 ± 3 days, term = 150 days gestation) were delivered by cesarean section and anesthetized with ketamine HCl (30 mg/kg iv) before rapid exsanguination. The ductus was divided into two 2-mm thick rings; each ring was mounted in a separate 20-ml organ culture bath and incubated in Krebs-bicarbonate solution (38°C) equilibrated with a gas mixture containing 5% CO2. The bath solution was exchanged at a rate of 10 ml/h (8, 10). The rings were stretched to a length that produced a maximal isometric contractile response to increases in oxygen tension (4.0 ± 0.1 mm) (4). The oxygen concentration in the baths was initially 5% while the rings were being mounted. The oxygen concentration was then switched to 80% O2, and the rings were incubated for 24 h in either 11.1 mM (2 mg/ml) glucose or 0.56 mM (0.1 mg/ml) glucose. Isometric tensions were recorded at the start of the experiment (5 min after mounting the ring) and after the 24-h incubation in the high oxygen environment. After incubation, the rings were embedded in Tissuetek (Miles) and frozen in liquid nitrogen for bioluminescence imaging of ATP and TUNEL staining.
Statistical analysis was performed by the appropriate Student’s t-test and by correlation and regression analysis. When more than one comparison was made, Bonferroni’s correction was used. Nonparametric data were compared with a Mann-Whitney U-test. Results are presented as means ± SD.
In the full-term baboon ductus, expression of our two surrogate markers of hypoxia (HIF1α and VEGF) increased (Fig. 1) and the average concentrations of ATP, glucose, and glycogen decreased (Figs. 2 and 3) after postnatal closure. At the same time, there was an increase in the number of TUNEL-positive cells in the ductus muscle media (Fig. 2D). The concentrations of ATP, glucose, and glycogen were related to the degree of ductus constriction (area of ductus lumen vs. ATP: r = 0.83, P < 0.08; vs. glucose: r = 0.96, P < 0.05; vs. glycogen: r = −0.96, P < 0.05, n = 5). We previously found that cell death (TUNEL-positive cells) in the full-term lamb ductus increased exponentially as the ductus constricted (16). In the full-term baboon ductus, there also was a significant relationship between the degree of ductus constriction and the logarithmic transformation of the number of TUNEL-positive cells (area of ductus lumen vs. TUNEL log10: r = −0.92, P < 0.05, n = 5).
Similarly, in the immature baboon ductus, HIF1α/VEGF expression increased and ATP concentrations decreased following delivery (Figs. 1 and 2A). Although the changes in HIF1α/VEGF expression and ATP concentrations were significantly related to the degree of ductus constriction (Figs. 1 and 2A) (area of ductus lumen vs. ATP: r = 0.77, P < 0.01, n = 16), both HIF1α/VEGF expression and ATP concentrations were still significantly altered after birth even when the immature ductus remained open and continued to have persistent luminal blood flow (Figs. 1 and 2A).
Average concentrations of glucose and glycogen decreased in the immature ductus after birth (Fig. 2, B and C). Although the presence of glucose depletion was significantly related to the degree of ductus constriction (Fig. 2B), the presence of glycogen depletion appeared to be independent of the absolute degree of constriction (Fig. 2C).
In the immature ductus, there was also a significant increase in the number of TUNEL-positive cells (P < 0.05) following delivery (Fig. 2D). Although the degree of TUNEL-positive staining was significantly related to the degree of ductus constriction (TUNEL log10 vs. area of ductus lumen: r = −0.69, P < 0.01, n = 16), extensive cell death (>15% TUNEL-positive cells) only occurred when the ductus was closed, and there was no Doppler flow through its lumen (Fig. 2D).
In the immature ductus, the degree of TUNEL staining was significantly related to the degree of ATP and glucose depletion (Fig. 4, A and B). ATP concentrations were directly related to tissue glucose concentrations (r = 0.52, P < 0.05, n = 16). Glycogen concentrations did not appear to be related to either the concentrations of ATP (data not shown) or the degree of TUNEL staining in the immature ductus (Fig. 4C).
We used ductus arteriosus from preterm lamb fetuses to determine whether the decrease in ATP concentrations in the immature open ductus could affect ductus contractility. We examined the contractile behavior of immature sheep ductus in vitro exposed to 80% O2 and two different concentrations of glucose. At low glucose concentrations, ATP concentrations were not maintained and fell to 31% of the starting (0 h) fetal ductus levels (Fig. 5). This is similar to the drop in ATP concentration observed in the immature newborn baboon ductus that remains open after birth in vivo (Fig. 2). There was no difference between the two experimental groups in the incidence of cell death (TUNEL staining) during the in vitro incubation (24 h). Ductus with normal concentrations of glucose and ATP contracted when exposed to 80% O2 for 24 h; in contrast, ductus with low glucose and low ATP failed to contract when exposed to 80% O2 (Fig. 5).
We found that after delivery, constriction of the full-term newborn baboon ductus arteriosus is associated with oxygen, glucose, glycogen, and ATP depletion and subsequent cell death in the ductus muscle media (Figs. 1 and 2). These findings confirm similar observations made in the full-term lamb ductus arteriosus (12). Prior in vitro studies have suggested that ductus muscle media cell death is due to ATP depletion and energy failure rather than to classical apoptotic mechanisms (8, 12). Cell death is most marked when both tissue oxygen and glucose reserves are severely depleted (8, 12).
The preterm ductus fails to develop the same degree of cell death after birth as the full-term ductus (2, 16). However, prior in vitro studies have shown that, if the preterm ductus develops the same degree of tissue hypoxia and glucose depletion as the full-term ductus, it is capable of developing a similar degree of severe ATP depletion and cell death (8, 12). The present studies are the first to demonstrate that constriction of the preterm ductus in vivo is associated with glucose and ATP depletion (Figs. 2 and 3) in addition to oxygen depletion (Fig. 1) in the ductus wall. In the preterm ductus, cell death (TUNEL staining) in vivo is significantly associated with ATP and glucose depletion just as it is in the full-term ductus (Fig. 4).
In the full-term newborn ductus arteriosus, severe oxygen, glucose, glycogen, and ATP depletion, cell death and tissue remodeling occur even before there is complete loss of luminal blood flow (11, 12); this is due to the marked increase in diffusion distances for oxygen and nutrients that occur after birth (11, 12). The present study demonstrates that the preterm ductus must completely obliterate its luminal blood flow before it develops the same degree of in vivo hypoxia, glucose depletion, and cell death as found in the full-term newborn ductus (Figs. 1 and 2).
In the immature ductus, tissue oxygen concentrations (Fig. 1) appear to be more significantly affected by postnatal constriction than are tissue glucose concentrations (Fig. 2B). In vitro studies have shown that, under similar conditions of hypoxia, the immature ductus has higher concentrations of tissue glucose than the full-term ductus (12). This appears to be due to increased glucose availability (12).
When ductus arteriosus (or other vessels) are studied in vitro, the primary substrate used during glycolysis and ATP generation is glucose, rather than glycogen (12, 13). The present in vivo findings support these in vitro findings. In the immature ductus, ATP concentrations are directly related to tissue glucose concentrations but not to glycogen concentrations. Similarly, cell death (TUNEL staining) is related to tissue glucose concentrations but not to glycogen concentrations in the immature ductus. Although partial constriction of the immature ductus was associated with glycogen depletion (Fig. 2C, immature newborn open), tight constriction produced regions of the ductus wall where glycogen concentrations were actually greater than those observed in the fetus (Fig. 3). We have previously shown that ductus rings, incubated in vitro under hypoxic conditions, develop both regions of glycogen depletion, as well as regions of glycogen surplus. Interestingly, the regions of glycogen surplus are more commonly seen in the immature than the mature ductus (12). We hypothesize that this adaptive mechanism could add to the ability of the immature ductus to tolerate episodes of hypoxia and nutrient shortage making it more resistant to developing postnatal cell death and permanent closure.
Cell death and remodeling of the preterm ductus only occur when there has been complete loss of luminal blood flow, with profound tissue anoxia and ATP depletion (Fig. 2D) (16). The current studies show that there are significant decreases in tissue oxygen (HIF1α/VEGF expression) (Fig. 1) and ATP concentrations (Fig. 2A) in the preterm ductus even when it continues to have persistent luminal blood flow after birth. Smooth muscle contractility is impaired by both hypoxia (10) and ATP depletion in the ductus (Fig. 4), as well as in other vessels (5, 6, 19). In the tightly constricted closed ductus, cell death and anatomic remodeling continue to hold the ductus closed despite the impaired muscle contractility. Metabolic depletion and diminished contractility still occur in the persistently patent ductus; however, cell death and tissue remodeling fail to develop and fail to obstruct the patent lumen.
These findings have important implications for clinical care. Prior studies have shown that indomethacin is most effective in closing the preterm PDA when it is given prophylactically within 24 h of birth (15). Its ability to produce ductus closure decreases with increasing postnatal age. We hypothesize that with increasing postnatal age, depletion of oxygen and ATP in the persistently patent ductus decreases its ability to constrict when exposed to indomethacin or other vasoconstrictors (1). Our current findings may explain why indomethacin becomes less effective in constricting the immature ductus with increasing postnatal age (15).
Supported by grants from U. S. Public Health Service (National Institutes of Health grants HL-46691, HL-56061, HL-52636 to the BPD Resource Center, and P51RR13986 for Primate Center facility support) and by a gift from the Gates Foundation. Seth Goldbarg is a research fellow with the Stanley J. Sarnoff Endowment for Cardiovascular Research.
The authors thank all the personnel that support the Broncho Pulmonary Dysplasia Resource Center: the Animal Husbandry Group led by Drs. D. Carey and M. Leland, the Neonatal Intensive Care Unit staff (H. Martin, D. Correll, S. Gomez, S. Ali, L. Kalisky, L. Nicley, R. Degan, S. Salazar), the Wilford Hall Medical Center neonatal fellows who assist in the care of the animals, and the University of Texas Health Science Center (San Antonio, TX) pathology staff (V. Winter, L. Buchanan, K. Symank, Y. Valdes and K. Mendoza) who perform necropsies.
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- Copyright © 2006 the American Physiological Society