LABORATORY INVESTIGATION CONGENITAL HEART DISEASE Persistent responsiveness of the neonatal ductus arteriosus in immature lambs: a possible cause for reopening of patent ductus arteriosus after indomethacin-induced closure RONALD 1. CLYMAN, M.D., DONNIE CAMPBELL, B.S.N., MICHAEL A. HEYMANN, M.D., AND FRANCOISE MAURAY, B.S. ABSTRACT Reopening of the ductus arteriosus after successful indomethacin-induced closure has become a major problem with indomethacin treatment. In full-term human newborns and lambs, the ductus behaves like ischemic tissue after its initial constriction. Its ability to continue to relax or contract depends on the amount of left-to-right shunt through the ductus lumen. To see if ductus constriction in preterm lambs would produce the same loss of ductus responsiveness, we delivered 42 lambs by cesarean section and ventilated them for 6.6 + 0.5( + SE) hr. We measured ductus arteriosus resistance and left-to-right shunt with the use of radionuclide-labeled microspheres. After the hemody- namic measurements were obtained, the ductus was studied in vitro. Immature lambs were more likely to have reactive ductus (after their initial ductus constriction) than were more mature lambs. This was due to a diminished degree of ductus constriction as well as persistence of ductus responsivenes in immature lambs when compared with more mature lambs. This persistence of ductus responsiveness in immature lambs after ductus constriction may account for the high reopening rate in preterm infants after successful indomethacin-induced closure. Circulation 71, No. 1, 141-145, 1985. SEVERAL recent controlled trials have demonstrated that indomethacin is effective in treating the patent ductus arteriosus (PDA)'-' in preterm infants. Howev- er, it has become apparent that once the ductus arterio- sus has been closed by indomethacin, it may reopen at a later date, with recurrence of the left-to-right shunt. The reported incidence of reopening of the ductus ar- teriosus after successful indomethacin treatment has varied from 20% to 100%.' 2,4, 6`0 These studies are difficult to compare since indomethacin was given at different postnatal ages to infants of different gesta- tional ages and in different amounts per dose. Howev- er, reopening of the ductus arteriosus did not appear to be associated with excessive fluid therapy before or after treatment7 or with low plasma concentrations of indomethacin during the initial therapy.'0 The mechanisms regulating patency and closure of From the Department of Pediatrics, Mt. Zion Hospital and Medical Center, and the Cardiovascular Research Institute and Department of Pediatrics. University of California, San Francisco. Supported by grants from the United States Public Health Service Program Project HL24056 and SCOR HL27356. Address for correspondence: Ronald Clyman, M.D., 1403 HSE, University of California, San Francisco, San Francisco, CA 94143. Received Aug. 17, 1984; accepted Sept. 20, 1984. Vol. 71, No. 1, January 1985 the ductus arteriosus are only partially understood. There appears to be a balance between the constricting effect of oxygen and the vasodilating effect of prosta- glandins. The marked sensitivity of the lamb ductus to prostaglandin E, (PGE,) suggests that this is the most important prostanoid for regulation of vessel patency in this species." However, within a few days after birth the relaxing effects of PGE, and hypoxia are lost and the ductus remains "irreversibly" closed there- after. 12-15 Fay and Cooke'3 have proposed that irrevers- ibility reflects a mechanical restraint imposed by cellu- lar necrosis, loss of an intact endothelium, and intermingling of cells from opposing walls such that there was no longer an anatomic lumen. Because ne- crosis and cell dislocation are limited to the central regions of the ductus, several authors have suggested that these processes result from interruption of luminal blood flow. 3' 16-1 We have previously observed a decrease in the abili- ty of the ductus arteriosus to dilate and contract that occurs within the first hours after postnatal ductus con- striction and before the loss of an anatomically patent lumen in human newborns and in full-term lambs.'2 141 by guest on June 17, 2018 http://circ.ahajournals.org/ Downloaded from
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LABORATORY INVESTIGATIONCONGENITAL HEART DISEASE
Persistent responsiveness of the neonatal ductusarteriosus in immature lambs: a possible cause forreopening of patent ductus arteriosus afterindomethacin-induced closureRONALD 1. CLYMAN, M.D., DONNIE CAMPBELL, B.S.N., MICHAEL A. HEYMANN, M.D.,AND FRANCOISE MAURAY, B.S.
ABSTRACT Reopening of the ductus arteriosus after successful indomethacin-induced closure hasbecome a major problem with indomethacin treatment. In full-term human newborns and lambs, theductus behaves like ischemic tissue after its initial constriction. Its ability to continue to relax or
contract depends on the amount of left-to-right shunt through the ductus lumen. To see if ductusconstriction in preterm lambs would produce the same loss of ductus responsiveness, we delivered 42lambs by cesarean section and ventilated them for 6.6 + 0.5( + SE) hr. We measured ductus arteriosusresistance and left-to-right shunt with the use of radionuclide-labeled microspheres. After the hemody-namic measurements were obtained, the ductus was studied in vitro. Immature lambs were more likelyto have reactive ductus (after their initial ductus constriction) than were more mature lambs. This was
due to a diminished degree of ductus constriction as well as persistence of ductus responsivenes inimmature lambs when compared with more mature lambs. This persistence of ductus responsiveness inimmature lambs after ductus constriction may account for the high reopening rate in preterm infantsafter successful indomethacin-induced closure.Circulation 71, No. 1, 141-145, 1985.
SEVERAL recent controlled trials have demonstratedthat indomethacin is effective in treating the patentductus arteriosus (PDA)'-' in preterm infants. Howev-er, it has become apparent that once the ductus arterio-sus has been closed by indomethacin, it may reopen ata later date, with recurrence of the left-to-right shunt.The reported incidence of reopening of the ductus ar-teriosus after successful indomethacin treatment hasvaried from 20% to 100%.' 2,4, 6`0 These studies aredifficult to compare since indomethacin was given atdifferent postnatal ages to infants of different gesta-tional ages and in different amounts per dose. Howev-er, reopening of the ductus arteriosus did not appear tobe associated with excessive fluid therapy before orafter treatment7 or with low plasma concentrations ofindomethacin during the initial therapy.'0The mechanisms regulating patency and closure ofFrom the Department of Pediatrics, Mt. Zion Hospital and Medical
Center, and the Cardiovascular Research Institute and Department ofPediatrics. University of California, San Francisco.
Supported by grants from the United States Public Health ServiceProgram Project HL24056 and SCOR HL27356.
Address for correspondence: Ronald Clyman, M.D., 1403 HSE,University of California, San Francisco, San Francisco, CA 94143.
Received Aug. 17, 1984; accepted Sept. 20, 1984.
Vol. 71, No. 1, January 1985
the ductus arteriosus are only partially understood.There appears to be a balance between the constrictingeffect of oxygen and the vasodilating effect of prosta-glandins. The marked sensitivity of the lamb ductus toprostaglandin E, (PGE,) suggests that this is the mostimportant prostanoid for regulation of vessel patencyin this species." However, within a few days afterbirth the relaxing effects of PGE, and hypoxia are lostand the ductus remains "irreversibly" closed there-after. 12-15 Fay and Cooke'3 have proposed that irrevers-ibility reflects a mechanical restraint imposed by cellu-lar necrosis, loss of an intact endothelium, andintermingling of cells from opposing walls such thatthere was no longer an anatomic lumen. Because ne-crosis and cell dislocation are limited to the centralregions of the ductus, several authors have suggestedthat these processes result from interruption of luminalblood flow. 3' 16-1We have previously observed a decrease in the abili-
ty of the ductus arteriosus to dilate and contract thatoccurs within the first hours after postnatal ductus con-striction and before the loss of an anatomically patentlumen in human newborns and in full-term lambs.'2
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This generalized loss of ductus responsiveness wasdirectly related to the degree of prior ductus constric-tion and the subsequent reduction in luminal bloodflow.12 The loss of ductus responsiveness was inde-pendent of arterial Pao2, pH, and PGE, concentra-tions."2 In addition, this loss of responsiveness pre-vented the ductus arteriosus from reopening once it hadconstricted. 12The purpose of the study reported here was to deter-
mine if premature lambs developed the same loss ofductus responsiveness after postnatal constriction asdid late-gestation lambs.Methods
Neonatal lambs - in vivo. Forty-two neonatal lambswere delivered by cesarean section at between 120 and 147 daysgestation (147 days was full term in our lambs) from time-datedpregnant ewes. In each neonate polyvinyl catheters wereplaced, via peripheral vessels, in the ascending aorta proximalto the ductus arteriosus and in the descending aorta, inferiorvena cava, left ventricle, pulmonary artery, and superior venacava as previously described. '1 Each fetus was intubated beforeseparation from the umbilical circulation. Fetuses were treatedwith 50 mg/kg surfactant lipid diluted in 0. 1 N NaCl by instilla-tion into the endotracheal tube. '9 The surfactant was isolatedfrom lungs from healthy adult ewes.19 We have previouslyshown that surfactant treatment of lambs has no effect on thebehavior of the ductus arteriosus in vivo or in vitro.19
After separation from the umbilical circulation the newbornlambs were paralyzed with 0.4 mg iv pancuronium and ventilat-ed with a timed-cycle pressure-limited infant ventilator. Theinitial ventilator settings were 28 cm H20 peak inspiratory pres-sure and 3 cm H20 positive end-expiratory pressure at a respira-tory rate of 40/min. The initial inspired gas was 100% oxygen.The ventilator settings and fraction of inspiratory oxygen werealtered to maintain a Pao, between 80 and 150 torr, Paco, lessthan 40 torr, and arterial pH between 7.35 and 7.45. Rectaltemperature was monitored and the temperature was maintainedat 38° to 390 C by a radiant warmer. Lambs were kept alive for atleast 3 hr after delivery (the mean survival time, 6.6 + 0.5 hr[-SE, n = 42j).We measured cardiac output and its distribution by injecting
radionuclide-labeled microspheres, 15 gm diameter, into theleft ventricle of each lamb. Reference samples were drawn fromthe ascending and descending aorta.20 Heparinized maternalblood was used to replace blood loss caused by sampling. Right-to-left ductal shunting was evaluated by microsphere injectionsinto the superior vena cava; there was no right-to-left ductalshunting in any of these animals. After the experiment eachanimal was autopsied to check catheter placement. The lungswere dissected, weighed, and processed for isotope counting aspreviously described.20 Left ventricular output and blood flowthrough the ductus arteriosus were calculated with use of theconcentration of microspheres in the reference sample, the totalnumber of microspheres recovered from the whole animal, andthe total number of microspheres in the lungs.'9 An assumedbronchial blood flow of 2% of left ventricular output was sub-tracted from the measured lung blood flows. 12 The microspheremeasurements were used to estimate left-to-right ductal shunts.Microsphere measurements were made at 1 hr after delivery andwere repeated at one or two hourly intervals as long as theanimal survived. Up to nine different microsphere injectionswere made in each animal.
Since no right-to-left ductal shunts were observed, systemic
blood flow equalled left ventricular output minus the left-to-right shunt through the ductus arteriosus. We estimated theresistance to flow across the ductus by dividing the mean de-scending aortic-pulmonary arterial pressure difference (torr) byductus arteriosus flow (liters/kg min). Systemic vascular resis-tance was estimated by dividing the mean descending aortic-inferior vena caval pressure difference by systemic blood flow.Hemodynamic measurements made during the last 2 to 3 hrbefore the animals were killed were averaged to determine thedegree of ductus constriction and reduction in luminal bloodflow in each animal.Lamb ductus in vitro. After completion of the study in
vivo, the lambs were killed rapidly by exsanguination. Theductus was dissected free from the adventitial tissue and dividedinto 1 mm thick rings that were placed in separate 150 ml organbaths kept in the dark within an enclosed box. The rings were
suspended between two stainless steel hooks in a modifiedKrebs Tris solution at 380 C.19, Isometric responses of cir-cumferential tension were measured by Grass FTO3C forcetransducers.
Each of the rings was stretched to an initial length that wouldresult in a maximal contractile response to increases in oxygen
tension. Initially the Po2 of the bath solution was maintained at20 to 25 torr and the rings were allowed to equilibrate for about1 hr until a steady tension developed. The bath solution was thenbubbled with 100% 02 (to a Po2 of 680 to 700 torr) until thetension achieved a new plateau. Indomethacin was added to thebath in a final concentration of 2 ,ug/ml (5.6 x 10- 6M) and therings were allowed to achieve a new increase in tension over thenext hour. A cumulative dose response to PGE2 was obtained inthe oxygen~ and indomethacin-contracted rings; we allowed thetension to achieve a new plateau before higher concentrations ofPGE2 were added. After the experiment the tissues were re-
moved from the baths and blotted dry and their wet weightswere determined. The tension developed in the ring was ex-
pressed as force per unit cross-sectional area (g/mm2).2To allow comparison with the activity in vitro of the 42
ductus that were isolated from newborn lambs after severalhours of ventilation. 38 fetuses (120 to 146 days gestation) were
delivered by cesarean section and killed before they took a
breath. Their ductus were divided into rings and suspended inthe organ baths by the same method as described for the new-born ductus.
Data analysis. All values are given as mean + SE. Signifi-cance was tested by a two-tailed unpaired t test, Fisher exact
test, or chi-squared analysis, when appropriate.
ResultsWe studied neonatal lambs of different gestational
ages to see how prematurity might alter the normalevents that occur in the ductus arteriosus after birth.Table 1 shows the degree of ductus arteriosus constric-tion that occurred in vivo in the 27 immature lambs(120 to 134 days gestation) and the 15 near-term lambs(135 to 147 days). We defined the degree of ductalconstriction as either "tight' or "moderate." In tight-ly constricted ductus there was both left-to-right shunt-ing that was less than 10% of the systemic blood flowand calculated ductus resistance greater than 1000 torr/litermin-kg. In moderately constricted ductus left-to-right shunting was greater than 10% of the systemicflow and resistance was less than 300 torr/liter-min-kg.
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Values are mean ± SE. Hemodynamic values for each lamb were obtained by averaging the measurements made during thelast 2 to 3 hr before they were killed. See text for definitions of tight and moderate constriction.Q -ctus left-to-right shunt through the ductus; Qys(emic = systemic blood flow; Rductus =resistance to flow across the ductus;
Rsystemic - septemic vascular resistance.
Immature lambs produced significantly weaker de-grees of ductus constriction than the more maturelambs: Only 26% (7/27) of the immature lambs devel-oped a tightly constricted ductus, whereas 67% (10/15)of the more mature animals were able to do so.
After the series of hemodynamic measurements, theanimals were killed to see how the effects of ductusconstriction in vivo altered the responsiveness of theductus arteriosus in vitro. With increasing degrees ofductus constriction in vivo, there was a significantlimitation of the ability of the more mature (> 135days) ductus to actively contract with oxygen and indo-methacin or to relax with PGE2 (table 2). Ductus isolat-ed from near-term lambs (' 135 days), which were
tightly constricted in vivo after several hours of venti-
lation, could generate only one-seventh the activetension in vitro (when exposed to oxygen and indo-methacin) of that generated by ductus isolated fromnear-term fetuses that were never ventilated and thathad patent ductus in vivo. In addition, PGE2 was only58% as effective in relaxing ductus isolated from near-
term newborn lambs (in which ductus were tightlyconstricted several hours after delivery) as it was inrelaxing those isolated from near-term fetal lambs(table 2).
In contrast, both the ability to actively contract as
well as to actively relax was significantly greater in theductus arteriosus of immature lambs (120 to 134 days)when compared with the ductus of more mature lambs(135 to 147 days) after similar degrees of constriction
TABLE 2Ductus arteriosus contractility: response in vitro vs that in vivo
Maximum PGE2 inhibition of active tensionActive tension (02 + indomethacin, g/mm2) (%)
Degree of ductus ___________________constriction in vivo 120-134 days 135-147 days 120-134 days 135-147 days
Values are mean + SE. See text for definition of moderate and tight constriction. Fetal ductus came from animals that werenever ventilated. All rings first were allowed to equilibrate in a low-Po2 environment (20 to 25 torr); steady-state low Po2 tensionswere: fetal, 120-134days = 3.14 ± 0.37 g/mm2, fetal, 135-147 days = 4.38 ± 0.29g/mm2; moderateconstriction, 120-134days = 3.00 ± 0.23 g/mm2, moderate constriction, 135-147 days = 2.86 ± 0.45 g/mm2; tight constriction, 120-134 days =
2.34 + 0.33 glmm2; tight constriction, 135-147 days = 2.70 ± 0.33 g/mm2. The oxygen + indomethacin tension is thedifference between the steady-state tensions at low Po2 and the tensions after the rings had been contracted with high Po2 (680 to700 torr) and indomethacin.
Ap < .05 when value for 135-147 day animals are compared with 120-134 day animals with a similar degree of ductusconstriction (fetal, moderate, or tight).
BRings from two lambs with tightly constricted ductus had such a minimal (0 and 0.2 g!mm2) active tension that PGE2inhibition could not be performed.
Vol. 71, No. 1, January 1985 143
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Values are mean ± SE. See footnote to table 2 for definitions.ED50 = effective dose for 50% inhibition of constriction.
in vivo (table 2). Isolated ductus from both immatureand mature fetuses that had never breathed had similarabilities to contract with oxygen and indomethacin andmaximally relax with PGE, (table 2). However, therewas no loss of the ability of PGE, to maximally relaxthe isolated ductus (studied in vitro) after increasingdegrees of postnatal constriction in vivo in the imma-ture animals as there was in the more mature lambs.Similarly, after each degree of postnatal constriction invivo, ductus from immature lambs retained twice thecontractile activity (to oxygen and indomethacin) ofductus from older lambs (table 2).As we observed previously," ductus from the most
immature fetuses were more sensitive to PGE, thanwere ductus from near-term fetuses (table 3). Althoughthe number of animals in each category was small,postnatal ductus constriction did not appear to alter thisdevelopmental shift in sensitivity.
DiscussionWe have previously observed a generalized loss of
ductus responsiveness and this occurred in the first
hours after ductus constriction and before the loss of ananatomically patent lumen.'2 This functional changeprobably reflects early ischemic damage to the innermuscle wall. As demonstrated in the present study,immature lambs are more likely to have reactive ductus(after their postnatal constriction) than are more ma-
ture lambs: (1) Immature lambs do not constrict theirductus as tightly as more mature lambs (table 1). Thisprobably results from an increased sensitivity of theimmature ductus to PGE, and higher circulating con-
centrations of PGE, in immature lambs.". 23 (2) Forthe same degree of ductus constriction in vivo, there isan increased persistence of ductus responsiveness inimmature lambs when compared with more maturelambs (table 2). The reason for this is unknown, butthis persistence of ductus responsiveness in immaturelambs after ductus constriction may account for thehigh reopening rate in preterm infants after successfulindomethacin-induced closure.From January 1, 1979 to December 31, 1983, 123
premature infants weighing less than 2500 g at birthwho were admitted before 24 hr of age to the NewbornIntensive Care Unit at Mount Zion Hospital and Medi-cal Center were treated with indomethacin for symp-
tomatic patent ductus arteriosus. The evidence of sig-
nificant left-to-right shunting in these infants has beenpreviously described' and includes increases in at leastthree of the following signs: precordial activity, theintensity and duration of the murmur, pulse pressure,
cardiothoracic ratio, pulmonary vascular engorge-
ment, and ratio of the left atrial diameter to the aorticroot diameter, and two-dimensional echocardiograph-ic and Doppler results. All infants received three oraldoses of indomethacin: the first was 0.2 mg/kg bodyweight, the second (given 12 hr later) was 0. 1 mg/kg,and the third (given 36 hr after the first) was 0.1mg/kg.
TABLE 4Characteristics of infants treated with indomethacin
Birthweight (g)Gestational age (wk + SE)Respiratory distress syndrome (%)Male/female (%)
Age when initial indomethacin treatment given(days)
Initial PDA closure rate with indomethacintreatment
Incidence of PDA recurrence after initial closureInterval between starting initial treatment andPDA reopening (days)
PDA closure rate with second indomethacintreatment
Eighty-seven percent of the infants treated with oralindomethacin had a positive initial response and nolonger had symptoms of hemodynamically significantpatent ductus arteriosus (table 4). There was no differ-ence in the initial response rate by birthweight or gesta-tional age. However, in 23% of those in whom theductus was initially closed by indomethacin, the duc-tus reopened and they again developed a symptomaticleft-to-right shunt requiring further treatment. The in-cidence of reopening was inversely related to the birth-weight: 33% of the infants with birthweights of lessthan 1000 g reopened their ductus after initial closure,while only 8% of infants with birthweights greater than1500 g reopened their ductus. Although indomethacinhas been found to be less effective in closing the ductusarteriosus when given later in the neonatal course,24this would not explain why infants weighing less than1000 g had such a high reopening rate.
Infants whose ductus reopened after initial closurestill appeared responsive to indomethacin. In 76% ofthe infants who were treated with indomethacin a sec-ond time (according to the same three-dose protocol)the ductus again closed (table 4). This is consistentwith our finding that the ductus arteriosus in immaturelambs maintains its ability to relax and contract afterpostnatal constriction.
We thank Dr. John Pike (Upjohn Co., Kalamazoo, Ml) forproviding us with PGE2 standards, and Mr. Carl McWatters andBruce Payne for their help in the preparation and analysis of themicrosphere data. We also thank Mr. Richard Truelove for hisskillful preparation of this manuscript and Dr. A. M. Rudolphfor his support during this project.
phylactic indomethacin therapy for patent ductus arteriosus in verylow birth weight infants. N Engl J Med 306: 506, 1982
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3. Yeh TF, Luken JA, Thalji A, Raval D, Carr I, Pildes RS: Intrave-nous indomethacin therapy in premature infants with persistentductus arteriosus a double blind controlled study. J Pediatr 98:137, 1981
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5. Yanagi RM, Wilson A, Newfeld EA. Axia KH, Hunt CE: Indo-methacin treatment for symptomatic patent ductus arteriosus: adouble blind controlled study. Pediatrics 67: 647, 1981
6. Ivey HH, Kattwinkel J, Park TS, Krovetz LJ: Failure of indometh-acin to close persistent ductus arteriosus in infants weighing under1000 grams. Br Heart J 41: 304, 1979
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19. Clyman RI, Jobe A, Heymann MA, Ikegami M, Roman C, PayneB, Mauray F: Increased shunt through the patent ductus arteriosusafter surfactant replacement therapy. J Pediatr 100: 101, 1982
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Vol. 71, No. 1, January 1985 145
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