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Studies on closure of the ductus arteriosus. XII. In utero effect of indomethacin and sodium salicylate in rats and rabbits
STUDIES EFFECT
ON CLOSURE OF
OF THE
INDOMETHACIN
AND
DUCTUS
ARTERIOSUS.
SODIUM
SALICYLATE
XII.
IN UTERO ---_---IN RATS AND
RABBITS
George
L.
Sharpe
1
, K. Sune
Larsson
and
Bertil
Thalme
Laboratory
Department
of Teratology, Karolinska Institutet, S-104 01 Stockholm and of Pediatrics, Karolinska Institutet, Huddinge Sjukhus, S-141 86 Huddinge, Sweden
ABSTRACT Administration of prostaglandin synthetase inhibitors to pregnant does and dams in late gestation was followed by in utero contraction of the fetal ductus arteriosus when -___-__studied by the whole-body freezing method. In the rat this contraction was well established within 6 h and persisted up to 36 h following 15 mg/kg indomethacin p.o. No effect was observed in the 18 d rat fetus but fetuses at 20 d and 22 d of gestation responded significantly to indomethacin. Doses of indomethacin approaching clinical usage (2.5 mg/kg) also caused a positive response in utero. The rat was found --:----to be sensitive also to sodium salicylate and in the rabbit both indomethacin and sodium salicylate were effective. Exposure in utero to prostaglandin synthetase inhibitors _-______ with resulting contraction of the ductus may seriously disturb cardiac function in the fetus.
from 1. Holder of a Fellowship Canadian Research Fund.
the
Queen
Elizabeth
II
This work was supported by grants No. 14X-993-09,10 and 17X-3912-03 from the Swedish Medical Research Council and from Stiftelsen Sigurd och Elsa Goljes Minne. Accepted April 10, 1975
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1975
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585
PRO’STAGLANDINS
XNTRODUCTION In vitro-studies (1) on full term lambs showing relaxa_-______ tion of ductal strips by prostaglandins (PGs) and ------in vivo studies (2) on newborn rats and rabbits demonstrating a reopening of the closing ductus arteriosus have indicated that PGs play a role in maintaining an open ductus prior to birth. It was further hypothesized that endogenous PGs inhibited the tendency of the ductus arteriosus to contract in response to oxygen. In a recent study, support for this view was added when ductal contraction occurred in utero in fetuses of dams given a PG synthetase inhibitor (indomethacin) 18 h before full term (3). The ductal response to oxygen appears in full only late in gestation (4,5) even if ___-__-_ in vitro studies have shown that the ductus arteriosus in several species contracted at earlier stages in gestation in response to chemical mediators such as acetylcholine, bradykinin and catecholamines (6,7,8,9). The aims of the present study were to; 1) further explore the time for onset and duration of demonstrated effect of indomethacin on intrauterine ductal constriction in the full term rat and 2) the effect at various gestational ages, 3) to evaluate the response to lower doses of indomethacin approaching clinical usage, 4) to study the effect of a second PG synthetase inhibitor (sodium salicylate) in the rat and 5) both inhibitors in a second species (rabbit). MATERIAL
AND
METHODS
Virgin Sprague-Dawley rats (Anticimex, Sollentuna) and virgin Havana rabbits (L. Sjdberg, Dlricehamn) were used in the experiment. The care of the animals and the methods of sacrifice and whole-body freezing have been described in detail in earlier reports (2,10,111. Newborn pups not frozen immediately at delivery were maintained in a 37'C incubator until sacrificed. Indomethacin (Merck, Sharp and Dohme) was given as a 1 mg/ml suspension insaline and sodium salicylate as a 100 mg/ml solution in saline. For each of the 5 parts of the study, 6 to 0 dams or 4 does were used to collect information on the fetuses and The mean inner ductal diameters of control and newborns. experimental groups were compared by Student's 't'test. All results from control groups (22 d rat) in parts l-4 were pooled to form a common reference group for statistical purposes. Appropriate controls of matching gestational age were used when studying rat fetuses before 22 d. of effect of indomethacin 1 Time of onset and duration _'________________,___,,____,-__________~~~~~~~~~~~~~~~~~-~ on intrauterine ductal size in the rat. ____________________c_________________ Rats received 15 mg/kg of indomethacin by gavage either on d 22 of pregnancy. 6, 24 or 36 h prior to sacrifice Litters from 2 dams receiving a corresponding volume of saline 24 h prior were included in a pooled control group.
586
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PROSTAGLANDINS
On surgical removal from the uterus the newborns were whole-body frozen within 20 set in a mixture of dry ice and 99.5 % ethanol while still in the intact amnion (time or at 15, 30 or 60 min following delivery.
0)
2 Influence of qestational on the effect of indo-I-----_---,_,-_--___-_-_-- aqe_________________-__---_ methacin on intrauterine ductal size in the rat. ____________________~~__~~~~~~~~~~~~~~~~~~_~___ Rats received either 15 mg/kg of indomethacin or a corresponding volume of saline for the control groups by gavage 18 h before sacrifice on either d 22, d 20 or d 18 of pregnancy. All pups were frozen immediately following delivery while still in the intact amnion (time 0). 3. Effect of various doses of indomethacin on __________________________~~~~~~~~~~~~~~~~~~~~~~~~~~ uterine ductal size in the rat. ____________________~~_~~~~~~~
intra-
Rats received either 2.5, 5 or 10 mg/kg of indomethacin by gavage 18 h before sacrifice on d 22 of pregnancy. Saline in a volume corresponding to the 10 mg/kg group was given to one dam whose litter was included in the control group. All pups were treated as in part 1. on intrauterine ductal 4. Effect of sodium salicylate ___________-_________---____________________~~~~~~~ size in the rat. _--__---___-___ Rats received either 50 or 100 mg/kg sodium salicylate S.C. 18 h prior to sacrifice on d 22 of pregnancy. Saline in a volume corresponding to 100 mg/kg sodium salicylate was given to one dam whose litter was included in the control group. All pups were treated as in part 1. 5. Effect of indomethacin and sodium salicylate on _______________________~~__~~___~~_~~~_~~~ ________i!?E”uterine ductal size in the rabbit. ____________________~~_~~~_~~~~~~ Rabbits received either 10 mg/kg indomethacin by gavage or 50 mg/kg sodium salicylate S.C. 18 h prior to sacrifice Litters from untreated does served on d 30 of pregnancy. All newborn pups were whole-body frozen immeas controls. diately following delivery (time 0) in liquid nitrogen. RESULTS Time of onset and duration of effect of indomethacin 1 _I____,___________-,_--___-_,-__-__-_,__~~~~~~~-~~~~~~~ on intrauterine ductal size in the rat (fiq._--1). ____________________~~_~~~~~~~~~~~~~~~~~~~ the ductus was open at delivery In the control animals, (time 01, but at 15 and 30 min a progressive contraction had occurred followed by a less rapid contraction at 60 min. oral dose of 15 mg/kg indomethacin, conAfter a single traction of the fetal ductus arteriosus was found to occur within 6 h and the effect persisted 36 h. At time 0 pups --__--__from dams receiving indomethacin 6, 24 or 36 h prior to decompared livery had significantly smaller ductal diameters to controls (6 h, p< 0.001; 24 h, p< 0.001; 36 h, p
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PROSTAGLANDINS
time groups compared to controls (6 h, p< 0.001; 24 h, p < 0.001; 36 h, p < 0.00.11. At 60 min no significant diffe-_-_---__ rence was present between control and experimental groups.
Sacritice delivery,
after min
I!!Ac !P
L
Hours prior to delivery on gestation d 22 of 15mg/kg indomethacin
c
62436
c
9
62436
7
9
C 62436
C
62436
p.0.
0
15
60
30
Fig. 1. Mean .nner ductal diameter (urn) + S. D. at delivery (0) and at 15, 30 and 60 min following 6, 24 or 36 h pretreatment with 15 mg/kg indomethacin p.o. (C denotes the pooled control group) prior to d 22 of gestation. Small numbers at bottom of each histogram denote number of rat specimens. 2 Influence of gestational on the effect of indo_'__---____----____-_-_____ age____________________-_methacin on intrauterine ductal size in the rat -~T-_~----'-‘--------~'--'--'-------~--~~---~~~~~~~~~ (fig 2) ____I___' Ductal contraction tjas present at delivery on d 22 and d 20 but not on d 18 when dams were given 15 mg/kg of indomethacin 18 h before. On d 22 the mean ductal diameter was 588 urn in the control and-'iZz urn in the indomethacin group.
588
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PROSTAGLANDINS
On ---a 20 the corresponding values were 484 and 211 Ym respec398 and tively, and for d la the values were d 18
d 20 d 22
.
I.!I T
a C 22 6
Daysof
dellvery, 15mglkg lndomethacln po. 18 h prior to deliver); Sacrlflce dellvery.
after mln
0
7
18
0
Fig. 2. Mean ductal diameter (pm) 2 S. D. at delivery following maternal pretreatment with 15 mg/kg indomethacin p.0. or vehicle (Cl at 18 h prior to d 22, 20 or 18 of gestation. Small numbers at bottom of each histogram, denote number of rat specimens.
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PROSTAGLANDINS
3. Effect of various doses of indomethacin on _____________~______~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ uterine ductal size in the rat Cfiq.____ 3). ____________________~~~~~~~~~~~~~~
intra-
A marked reduction in ductal size was present at delivery in fetuses of dams given 2.5, 5 or 10 mg/kg of indomethacin 18 h prior to delivery. At time 0 all pups from indometha_____W___) tin treated dams had a significantly smaller inner ductal diameter compared to controls (2.5 mg/kg, p< 0.001; 5 mg/kg, p < 0.001; 10 mg/kg, p < 0.001). Pups from the 10 mg/kg group had a more marked contraction than those in the 5 or 2.5 mg/kg groups (p < 0.001). At 15 min and at 30 min after --___---delivery all indomethacin treated pups stili';ai-a-significant reduction in ductal diameter compared to the controls (at 15 min: 2.5 mg/kg, p< 0.01; 5 mg/kg, p< 0.001; 10 mg/kg, p < 0.001. At 30 min: 2.5 mg/kg, p< 0.001; 5 mg/kg, p< 0.001; 10 mg/kg, p < 0.001). At 60 min however no significant diffe_-_-____rence was present between indomethacin and control groups.
L
Indomcthacin, mglkg p-o. 18 h prror to dellvery on gestation d 22 Sacriftce dellvery.
after mln
c 2.5 5
0
10
c 2.5 5
10
C 2.5
15
5
30
x)
C 2.5 5 10
60
(urn) + S. D. at delivery Fig. 3. Mean inner ductal diameter maternal pretreatment (0) and at 15, 30 and 60 min following p.o. (C denotes the with 2.5, 5 or 10 mg/kg indomethacin pooled control group) prior to d 22 of gestation. Small numbers at bottom of each histogram denote number of rat specimens.
590
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4 Effect of sodium salicxlate on intrauterine ductal _I________,_,_,_,_,_,-___-____________________------size in the rat (fig. 4). ____________________---Ductal contraction was present at delivery in full term fetuses of dams given either 50 or 100 mg/kg of sodium salicylate 18 h before. At time 0 and ______ 15 min pups from _________ sodium salicvlate treated dams had siqnificant contraction of the ductui compared to controls (50 mg/kg, p< 0.001; 100 mg/kg, p < 0.001). The difference was maintained at 30 min (50 mg/kg, p< 0.01; 100 mg/kg, p< 0.001). At gc min ______ ___W___ only the pups in the 100 mg/kg group had a significantly smaller ductal diameter (p< 0.001) compared to controls and pups in the 50 mg/kg group.
F 6001
L 5 6
No-Salicylate. mglkg s.c 18 h prior to delivery on gestation Sacrifice delivery.
d 22
after mln
5
C5OUXJ
0
T
hb 9 6
c
50100
30
T
76
C5OlOO
60
Fig. 4. Mean inner ductal diameter (pm) +- S. D. at delivery and at 15, 30 and 60 min following maternal pretreatment with 50 or 100 mg/kg sodium salicylate S.C. (C denotes the pooled control group) prior to d 22 of gestation. Small numbers at bottom of each histogram denote number of rat specimens.
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PROSTAGLANDINS
5. Effect of indomethacin and sodium salicylate ____________________~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ intrauterine ductal size in the rabbit (fig. ____________________~~~~~~~~~~~~~~~~~~~~~~~~~~
on 5).
Ductal contraction was present at delivery in the full term pups of pregnant does given either 10 mg/kg indomethacin or 50 mg/kg sodium salicylate 18 h prior to delivery on d 30. The ductal diameter was reduced to about one-fifth of that in the untreated control animals (P < 0.001).
Untreated
Sacrifice delivery.
after min
control
Indomethacin,lOmg/kg 18 h prior to delivery on gestation d 30
p.o
Na-Salicylate,5Omg/kg 18 h prior to delivery on gestation d 30
0
0
0
s.c
5. Mean ductal diameter (urn) + S.D. at delivery following 10 mg/kg indomethacin p.o. or 50 mg/kg sodium salicylate S.C. 18 h prior to d 30 of gestation. Newborns from untreated does served as controls. Small numbers at bottom of each histogram denote number of rabbit specimens.
Fig.
DISCUSSION
The reliability of the whole-body freezing method for studies of minute deviations from the normal closure process has been discussed by Hdrnblad (12). He concluded that this method gives a clear picture of the ductus at delivery exhibiting characteristics of vessels in vivo, i.e.: straight ------elastic lamella and a single endothelial layer. Furthermore, direct measurements of the ductus in live lamb and guinea pigs (8) are in accordance with the values obtained by Hdrnblad using the whole-body freezing technique. Since the ductus can not be considered to freeze instantaneously,
592
APRIL
1975 VOL. 9 NO. 4
PROSTAGLANDINS
the question can be raised whether or not this technique is appropriate for studies on the effects of prostaglandin synthetase inhibitors with reference to the physiological situation in utero.The visualization of the ductus in a -------presumed in vivo condition and the use of control animals -__-___ made it possible for a cautious interpretation of the finthe ductal closure rates of the control dings. Furthermore animals in the present study were' in good agreement with earlier results in the rat and rabbit (11). Bearing this in mind the results of the present investigation are in agreement with our previous demonstration of a marked interference with prepartum patency of the ductus arteriosus by PG synthetase inhibitors (3). Thus the ductal constriction was pronounced, long lasting and restricted to a definitive time period in the latter part of gestation. It is important to note that indomethacin was effective at clinical dose levels and that sodium salicylate had the same effect in the rat and in the rabbit. Indomethacin caused a marked, prompt constriction of the fetal ductus within 6 h, lasting at least up to 36 h after a single dose in the rat. The greater variation in ductal diameter in the 36 h group might indicate a weakening of the effect at this time. This long lasting effect on the rat and rabbit fetuses might be related to differences in tissue distribution of the drug in mother and fetus with a slower elimination in the fetus. Furthermore a recent report on indomethacin pharmacokinetics during pregnancy has showed higher concentrations in the human newborn relative to maternal levels after prepartum ingestion of the drug (13). It might therefore be expected that with repeated doses on a clinical basis, fetal levels of indomethacin may reach considerable concentrations. The demonstrated effectiveness of indomethacin from 6 to 36 h justifies the use of the 18 h pretreatment in the major part of our experiments. In a previous paper describing the effect of indomethacin on ductal patency, a dose of 15 mg/kg was used (3). In the present study the rat proved to be very sensitive to smaller doses of indomethacin. The range of 2.5 mg/kg is probably comparable to the dose received in clinical situations. In studies with the use of indomethacin to inhibit hypertonic saline induced abortions in humans (14) and to prevent normal term labour in rhesus monkeys (15) a dose approximating 2 mg/kg was found to be effective. A gestational variation in sensitivity towards indomethatin was clearly demonstrated in the present study. While the 18 d rat fetus showed no reactivity, the 20 d rat fetus displayed about half the sensitivity of that seen in the full term 22 d fetus. Sensitivity to oxygen is aquired later in gestation in other species, even if the ductus contracts in early gestation in response to acetylcholine, bradykinin and catecholamine (4-9). It seems likely that a depletion of endogenous PG would facilitate normal ductal closure. The
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presence of PG degrading enzymes in fetal lung up to 1000 times that of the adult rat may have such a function(l6). In our model local PGs were presumably depleted by the use of the PG synthetase inhibitors, indomethacin and sodium salicylate. Sodium salicylate, also effective in causing ductal contraction in utero is rated less potent than -------acetylsalicylic acid as an inhibitor of PG synthetase (17). Other inhibitors of PG synthetase as acetylsalicylic acid, paracetamol or naproxene (18) might conceivably reproduce this effect in utero. There has been at least one documen--_----ted case of ________ in utero ductal closure in man associated with salicylate exposure at 35 w of pregnancy (19). Further documentation of cases with premature closure of the ductus arteriosus is being collected by Kohler who himself has noted 3 cases in stillborn infants at autopsy (20, 21). The positive findings in the rabbit ductus using both indomethatin and sodium salicylate increase the probability that other species including man would show similar effects. Elliott and colleagues recently reported on improvement in arterial oxygen saturation after PG El infusion in two infants with severe congenital heart disease and dependent upon a patent ductus for pulmonary perfusion. The administration of indomethacin to one infant was associated with a drop in arterial saturation which may have resulted from ductus narrowing (22). This observation is in accordance with the contraction of the ductus arteriosus ig utero obtained by giving indomethacin to dams prior to ----operative delivery (3). We have previously remarked on the possible consequences of intrauterine ductal closure on cardiovascular performana contracted ductal ce at birth (3). In these experiments, diameter in utero would retard significant blood flow through ________ this vessel and cause a changed hemodynamic situation with an increased load on the heart. ACKNOWLEDGEMENT We wish to acknowledge and thank Marianne Kjellberg and Inger Unnerstad for valuable technical assistance; and Ulla Kudryk for preparation of the manuscript and typing. REFERENCES 1.
2.
3.
594
of the ducF. and P. M. Olley. The response Coceani, tus arteriosus to prostaglandins, Canad. J. Physiol. Pharmacol. 51:220, 1973. Sharpe, G. L. and K. S. Larsson. Studies on closure of In vivo effect of prostathe ductus arteriosus. X. ------Prostaglandins (submitted). glandin, Sharpe, G. L., B. Thalme and K. S. Larsson. Studies on closure of the ductus arteriosus. XI. Ductal closure in utero by a prostaglandin synthetase inhibitor, -----_-Prostaglandins, 8:363, 1974.
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4.
5.
6.
7.
8. 9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
Ikeda, M., E. H. Rubinstein and R. R. Sonnenschein. Development of the 02 induced contractions in the ductus arteriosus of the guinea pig, Experientia 29:445, 1973. McMurphy, D. M., M. A. Heymann, A. M. Rudolph and K. L. Melmon. Developmental changes in constriction of the ductus arteriosus: Responses to oxygen and vasoactive agents in the isolated ductus arteriosus of the fetal lamb, Pediat. Res. 6:231, 1972. Boreus, L. O., T. Malmfors, D. M. McMurphy and L. Olson. .Demonstration of adrenergic receptor function and innervation in the ductus arteriosus of the human fetus, Acta physiol. stand. 77~316, 1969. McMurphy, D. M. and L. 0. Boreus. Studies on the pharmacology of the perfused human fetal ductus arteriosus, Amer. J. Obstet. Gynec. 109:937, 1971. KovalEl'k, V. The response of the isolated ductus arteriosus to oxygen and anoxia, J. Physiol. 169:185, 1963. Aronson, S., G. Gennser, C. H. Owman and N.-O. SjiSberg. Innervation and contractile response of the human ductus arteriosus, Europ. J. of Pharmacol. 11:178, 1970. Hbrnblad, P. Y. and K. S. Larsson. Studies on closure of the ductus arteriosus. I. Whole-body freezing as improvement of fixation procedures, Cardiologia 51:231, 1967. HBrnblad, P. Y. Studies on closure of the ductus arteriosus. III. Species differences in closure rate and morphology, Cardiologia 51:262, 1967. Hbrnblad, P. Y. Experimental studies on closure of the ductus arteriosus utilizing whole-body freezing, Acta Paediat. Stand. supp. 190, 1969. Traeger, A., H. Ndschel and J. Zaumseil. Zur Pharmakokinetik von Indomethacin bei Schwangeren, Kreissenden und deren Neugeborenen, Zbl. Gynlk. 95:635, 1973. Waltman, R., V. Tricomi and A. Palav. Aspirin and Effect on instillation/abortion time of Indomethacin. mid-trimester hypertonic saline induced abortion, Prostaglandins 3:47, 1973. Novy, M. J., M. J. Cook and L. Manaugh. Indomethacin block of normal onset of partuition in primates, Am. J. Obstet. Gynecol. 118:412, 1974. Prostaglandins during Pace-Asciak, C. and D. Miller. development. I. Age-dependent activity profiles of prostaglandin 15-hydroxydehydrogenase and 13, 14reductase in lung tissue from late prenatal, early postnatal and adult rats, Prostaglandins 4:351, 1973. of prostaglandin synthesis as a Vane, J. R. Inhibition mechanism of action for aspirin-like drugs, Nature New Biol. 231:232, 1971. bioFlower, R. J. Drugs which inhibit prostaglandin synthesis, Pharmacol. Rev. 26:33, 1974.
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19.
20.
21. 22.
596
Arcilla, R. A., 0. G. Thilenius and K. Ranniger. Congestive heart failure from suspected ductal closure 75:74, 1969. in utero J. of Pediat. __-_--__' cardiac failure associated Kohler, H. G. Intrauterine with premature closure of the ductus arteriosus. Arch. 1967. Dis. Child. 42:335, communication. Kohler, H. G. Personal Elliott, R. B., M. B. Starling and J. M. Neutze. Medical manipulation of the ductus arteriosus. Lancet I:140, 1975.
APRIL
1975
VOL. 9 NO. 4
ON CLOSURE OF
OF THE
INDOMETHACIN
AND
DUCTUS
ARTERIOSUS.
SODIUM
SALICYLATE
XII.
IN UTERO ---_---IN RATS AND
RABBITS
George
L.
Sharpe
1
, K. Sune
Larsson
and
Bertil
Thalme
Laboratory
Department
of Teratology, Karolinska Institutet, S-104 01 Stockholm and of Pediatrics, Karolinska Institutet, Huddinge Sjukhus, S-141 86 Huddinge, Sweden
ABSTRACT Administration of prostaglandin synthetase inhibitors to pregnant does and dams in late gestation was followed by in utero contraction of the fetal ductus arteriosus when -___-__studied by the whole-body freezing method. In the rat this contraction was well established within 6 h and persisted up to 36 h following 15 mg/kg indomethacin p.o. No effect was observed in the 18 d rat fetus but fetuses at 20 d and 22 d of gestation responded significantly to indomethacin. Doses of indomethacin approaching clinical usage (2.5 mg/kg) also caused a positive response in utero. The rat was found --:----to be sensitive also to sodium salicylate and in the rabbit both indomethacin and sodium salicylate were effective. Exposure in utero to prostaglandin synthetase inhibitors _-______ with resulting contraction of the ductus may seriously disturb cardiac function in the fetus.
from 1. Holder of a Fellowship Canadian Research Fund.
the
Queen
Elizabeth
II
This work was supported by grants No. 14X-993-09,10 and 17X-3912-03 from the Swedish Medical Research Council and from Stiftelsen Sigurd och Elsa Goljes Minne. Accepted April 10, 1975
PROSTAGLANDINS APRIL
1975
VOL. 9 NO. 4
585
PRO’STAGLANDINS
XNTRODUCTION In vitro-studies (1) on full term lambs showing relaxa_-______ tion of ductal strips by prostaglandins (PGs) and ------in vivo studies (2) on newborn rats and rabbits demonstrating a reopening of the closing ductus arteriosus have indicated that PGs play a role in maintaining an open ductus prior to birth. It was further hypothesized that endogenous PGs inhibited the tendency of the ductus arteriosus to contract in response to oxygen. In a recent study, support for this view was added when ductal contraction occurred in utero in fetuses of dams given a PG synthetase inhibitor (indomethacin) 18 h before full term (3). The ductal response to oxygen appears in full only late in gestation (4,5) even if ___-__-_ in vitro studies have shown that the ductus arteriosus in several species contracted at earlier stages in gestation in response to chemical mediators such as acetylcholine, bradykinin and catecholamines (6,7,8,9). The aims of the present study were to; 1) further explore the time for onset and duration of demonstrated effect of indomethacin on intrauterine ductal constriction in the full term rat and 2) the effect at various gestational ages, 3) to evaluate the response to lower doses of indomethacin approaching clinical usage, 4) to study the effect of a second PG synthetase inhibitor (sodium salicylate) in the rat and 5) both inhibitors in a second species (rabbit). MATERIAL
AND
METHODS
Virgin Sprague-Dawley rats (Anticimex, Sollentuna) and virgin Havana rabbits (L. Sjdberg, Dlricehamn) were used in the experiment. The care of the animals and the methods of sacrifice and whole-body freezing have been described in detail in earlier reports (2,10,111. Newborn pups not frozen immediately at delivery were maintained in a 37'C incubator until sacrificed. Indomethacin (Merck, Sharp and Dohme) was given as a 1 mg/ml suspension insaline and sodium salicylate as a 100 mg/ml solution in saline. For each of the 5 parts of the study, 6 to 0 dams or 4 does were used to collect information on the fetuses and The mean inner ductal diameters of control and newborns. experimental groups were compared by Student's 't'test. All results from control groups (22 d rat) in parts l-4 were pooled to form a common reference group for statistical purposes. Appropriate controls of matching gestational age were used when studying rat fetuses before 22 d. of effect of indomethacin 1 Time of onset and duration _'________________,___,,____,-__________~~~~~~~~~~~~~~~~~-~ on intrauterine ductal size in the rat. ____________________c_________________ Rats received 15 mg/kg of indomethacin by gavage either on d 22 of pregnancy. 6, 24 or 36 h prior to sacrifice Litters from 2 dams receiving a corresponding volume of saline 24 h prior were included in a pooled control group.
586
APRIL
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PROSTAGLANDINS
On surgical removal from the uterus the newborns were whole-body frozen within 20 set in a mixture of dry ice and 99.5 % ethanol while still in the intact amnion (time or at 15, 30 or 60 min following delivery.
0)
2 Influence of qestational on the effect of indo-I-----_---,_,-_--___-_-_-- aqe_________________-__---_ methacin on intrauterine ductal size in the rat. ____________________~~__~~~~~~~~~~~~~~~~~~_~___ Rats received either 15 mg/kg of indomethacin or a corresponding volume of saline for the control groups by gavage 18 h before sacrifice on either d 22, d 20 or d 18 of pregnancy. All pups were frozen immediately following delivery while still in the intact amnion (time 0). 3. Effect of various doses of indomethacin on __________________________~~~~~~~~~~~~~~~~~~~~~~~~~~ uterine ductal size in the rat. ____________________~~_~~~~~~~
intra-
Rats received either 2.5, 5 or 10 mg/kg of indomethacin by gavage 18 h before sacrifice on d 22 of pregnancy. Saline in a volume corresponding to the 10 mg/kg group was given to one dam whose litter was included in the control group. All pups were treated as in part 1. on intrauterine ductal 4. Effect of sodium salicylate ___________-_________---____________________~~~~~~~ size in the rat. _--__---___-___ Rats received either 50 or 100 mg/kg sodium salicylate S.C. 18 h prior to sacrifice on d 22 of pregnancy. Saline in a volume corresponding to 100 mg/kg sodium salicylate was given to one dam whose litter was included in the control group. All pups were treated as in part 1. 5. Effect of indomethacin and sodium salicylate on _______________________~~__~~___~~_~~~_~~~ ________i!?E”uterine ductal size in the rabbit. ____________________~~_~~~_~~~~~~ Rabbits received either 10 mg/kg indomethacin by gavage or 50 mg/kg sodium salicylate S.C. 18 h prior to sacrifice Litters from untreated does served on d 30 of pregnancy. All newborn pups were whole-body frozen immeas controls. diately following delivery (time 0) in liquid nitrogen. RESULTS Time of onset and duration of effect of indomethacin 1 _I____,___________-,_--___-_,-__-__-_,__~~~~~~~-~~~~~~~ on intrauterine ductal size in the rat (fiq._--1). ____________________~~_~~~~~~~~~~~~~~~~~~~ the ductus was open at delivery In the control animals, (time 01, but at 15 and 30 min a progressive contraction had occurred followed by a less rapid contraction at 60 min. oral dose of 15 mg/kg indomethacin, conAfter a single traction of the fetal ductus arteriosus was found to occur within 6 h and the effect persisted 36 h. At time 0 pups --__--__from dams receiving indomethacin 6, 24 or 36 h prior to decompared livery had significantly smaller ductal diameters to controls (6 h, p< 0.001; 24 h, p< 0.001; 36 h, p
APRIL
1975
VOL. 9 NO. 4
587
PROSTAGLANDINS
time groups compared to controls (6 h, p< 0.001; 24 h, p < 0.001; 36 h, p < 0.00.11. At 60 min no significant diffe-_-_---__ rence was present between control and experimental groups.
Sacritice delivery,
after min
I!!Ac !P
L
Hours prior to delivery on gestation d 22 of 15mg/kg indomethacin
c
62436
c
9
62436
7
9
C 62436
C
62436
p.0.
0
15
60
30
Fig. 1. Mean .nner ductal diameter (urn) + S. D. at delivery (0) and at 15, 30 and 60 min following 6, 24 or 36 h pretreatment with 15 mg/kg indomethacin p.o. (C denotes the pooled control group) prior to d 22 of gestation. Small numbers at bottom of each histogram denote number of rat specimens. 2 Influence of gestational on the effect of indo_'__---____----____-_-_____ age____________________-_methacin on intrauterine ductal size in the rat -~T-_~----'-‘--------~'--'--'-------~--~~---~~~~~~~~~ (fig 2) ____I___' Ductal contraction tjas present at delivery on d 22 and d 20 but not on d 18 when dams were given 15 mg/kg of indomethacin 18 h before. On d 22 the mean ductal diameter was 588 urn in the control and-'iZz urn in the indomethacin group.
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On ---a 20 the corresponding values were 484 and 211 Ym respec398 and tively, and for d la the values were d 18
d 20 d 22
.
I.!I T
a C 22 6
Daysof
dellvery, 15mglkg lndomethacln po. 18 h prior to deliver); Sacrlflce dellvery.
after mln
0
7
18
0
Fig. 2. Mean ductal diameter (pm) 2 S. D. at delivery following maternal pretreatment with 15 mg/kg indomethacin p.0. or vehicle (Cl at 18 h prior to d 22, 20 or 18 of gestation. Small numbers at bottom of each histogram, denote number of rat specimens.
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3. Effect of various doses of indomethacin on _____________~______~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ uterine ductal size in the rat Cfiq.____ 3). ____________________~~~~~~~~~~~~~~
intra-
A marked reduction in ductal size was present at delivery in fetuses of dams given 2.5, 5 or 10 mg/kg of indomethacin 18 h prior to delivery. At time 0 all pups from indometha_____W___) tin treated dams had a significantly smaller inner ductal diameter compared to controls (2.5 mg/kg, p< 0.001; 5 mg/kg, p < 0.001; 10 mg/kg, p < 0.001). Pups from the 10 mg/kg group had a more marked contraction than those in the 5 or 2.5 mg/kg groups (p < 0.001). At 15 min and at 30 min after --___---delivery all indomethacin treated pups stili';ai-a-significant reduction in ductal diameter compared to the controls (at 15 min: 2.5 mg/kg, p< 0.01; 5 mg/kg, p< 0.001; 10 mg/kg, p < 0.001. At 30 min: 2.5 mg/kg, p< 0.001; 5 mg/kg, p< 0.001; 10 mg/kg, p < 0.001). At 60 min however no significant diffe_-_-____rence was present between indomethacin and control groups.
L
Indomcthacin, mglkg p-o. 18 h prror to dellvery on gestation d 22 Sacriftce dellvery.
after mln
c 2.5 5
0
10
c 2.5 5
10
C 2.5
15
5
30
x)
C 2.5 5 10
60
(urn) + S. D. at delivery Fig. 3. Mean inner ductal diameter maternal pretreatment (0) and at 15, 30 and 60 min following p.o. (C denotes the with 2.5, 5 or 10 mg/kg indomethacin pooled control group) prior to d 22 of gestation. Small numbers at bottom of each histogram denote number of rat specimens.
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4 Effect of sodium salicxlate on intrauterine ductal _I________,_,_,_,_,_,-___-____________________------size in the rat (fig. 4). ____________________---Ductal contraction was present at delivery in full term fetuses of dams given either 50 or 100 mg/kg of sodium salicylate 18 h before. At time 0 and ______ 15 min pups from _________ sodium salicvlate treated dams had siqnificant contraction of the ductui compared to controls (50 mg/kg, p< 0.001; 100 mg/kg, p < 0.001). The difference was maintained at 30 min (50 mg/kg, p< 0.01; 100 mg/kg, p< 0.001). At gc min ______ ___W___ only the pups in the 100 mg/kg group had a significantly smaller ductal diameter (p< 0.001) compared to controls and pups in the 50 mg/kg group.
F 6001
L 5 6
No-Salicylate. mglkg s.c 18 h prior to delivery on gestation Sacrifice delivery.
d 22
after mln
5
C5OUXJ
0
T
hb 9 6
c
50100
30
T
76
C5OlOO
60
Fig. 4. Mean inner ductal diameter (pm) +- S. D. at delivery and at 15, 30 and 60 min following maternal pretreatment with 50 or 100 mg/kg sodium salicylate S.C. (C denotes the pooled control group) prior to d 22 of gestation. Small numbers at bottom of each histogram denote number of rat specimens.
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5. Effect of indomethacin and sodium salicylate ____________________~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ intrauterine ductal size in the rabbit (fig. ____________________~~~~~~~~~~~~~~~~~~~~~~~~~~
on 5).
Ductal contraction was present at delivery in the full term pups of pregnant does given either 10 mg/kg indomethacin or 50 mg/kg sodium salicylate 18 h prior to delivery on d 30. The ductal diameter was reduced to about one-fifth of that in the untreated control animals (P < 0.001).
Untreated
Sacrifice delivery.
after min
control
Indomethacin,lOmg/kg 18 h prior to delivery on gestation d 30
p.o
Na-Salicylate,5Omg/kg 18 h prior to delivery on gestation d 30
0
0
0
s.c
5. Mean ductal diameter (urn) + S.D. at delivery following 10 mg/kg indomethacin p.o. or 50 mg/kg sodium salicylate S.C. 18 h prior to d 30 of gestation. Newborns from untreated does served as controls. Small numbers at bottom of each histogram denote number of rabbit specimens.
Fig.
DISCUSSION
The reliability of the whole-body freezing method for studies of minute deviations from the normal closure process has been discussed by Hdrnblad (12). He concluded that this method gives a clear picture of the ductus at delivery exhibiting characteristics of vessels in vivo, i.e.: straight ------elastic lamella and a single endothelial layer. Furthermore, direct measurements of the ductus in live lamb and guinea pigs (8) are in accordance with the values obtained by Hdrnblad using the whole-body freezing technique. Since the ductus can not be considered to freeze instantaneously,
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the question can be raised whether or not this technique is appropriate for studies on the effects of prostaglandin synthetase inhibitors with reference to the physiological situation in utero.The visualization of the ductus in a -------presumed in vivo condition and the use of control animals -__-___ made it possible for a cautious interpretation of the finthe ductal closure rates of the control dings. Furthermore animals in the present study were' in good agreement with earlier results in the rat and rabbit (11). Bearing this in mind the results of the present investigation are in agreement with our previous demonstration of a marked interference with prepartum patency of the ductus arteriosus by PG synthetase inhibitors (3). Thus the ductal constriction was pronounced, long lasting and restricted to a definitive time period in the latter part of gestation. It is important to note that indomethacin was effective at clinical dose levels and that sodium salicylate had the same effect in the rat and in the rabbit. Indomethacin caused a marked, prompt constriction of the fetal ductus within 6 h, lasting at least up to 36 h after a single dose in the rat. The greater variation in ductal diameter in the 36 h group might indicate a weakening of the effect at this time. This long lasting effect on the rat and rabbit fetuses might be related to differences in tissue distribution of the drug in mother and fetus with a slower elimination in the fetus. Furthermore a recent report on indomethacin pharmacokinetics during pregnancy has showed higher concentrations in the human newborn relative to maternal levels after prepartum ingestion of the drug (13). It might therefore be expected that with repeated doses on a clinical basis, fetal levels of indomethacin may reach considerable concentrations. The demonstrated effectiveness of indomethacin from 6 to 36 h justifies the use of the 18 h pretreatment in the major part of our experiments. In a previous paper describing the effect of indomethacin on ductal patency, a dose of 15 mg/kg was used (3). In the present study the rat proved to be very sensitive to smaller doses of indomethacin. The range of 2.5 mg/kg is probably comparable to the dose received in clinical situations. In studies with the use of indomethacin to inhibit hypertonic saline induced abortions in humans (14) and to prevent normal term labour in rhesus monkeys (15) a dose approximating 2 mg/kg was found to be effective. A gestational variation in sensitivity towards indomethatin was clearly demonstrated in the present study. While the 18 d rat fetus showed no reactivity, the 20 d rat fetus displayed about half the sensitivity of that seen in the full term 22 d fetus. Sensitivity to oxygen is aquired later in gestation in other species, even if the ductus contracts in early gestation in response to acetylcholine, bradykinin and catecholamine (4-9). It seems likely that a depletion of endogenous PG would facilitate normal ductal closure. The
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presence of PG degrading enzymes in fetal lung up to 1000 times that of the adult rat may have such a function(l6). In our model local PGs were presumably depleted by the use of the PG synthetase inhibitors, indomethacin and sodium salicylate. Sodium salicylate, also effective in causing ductal contraction in utero is rated less potent than -------acetylsalicylic acid as an inhibitor of PG synthetase (17). Other inhibitors of PG synthetase as acetylsalicylic acid, paracetamol or naproxene (18) might conceivably reproduce this effect in utero. There has been at least one documen--_----ted case of ________ in utero ductal closure in man associated with salicylate exposure at 35 w of pregnancy (19). Further documentation of cases with premature closure of the ductus arteriosus is being collected by Kohler who himself has noted 3 cases in stillborn infants at autopsy (20, 21). The positive findings in the rabbit ductus using both indomethatin and sodium salicylate increase the probability that other species including man would show similar effects. Elliott and colleagues recently reported on improvement in arterial oxygen saturation after PG El infusion in two infants with severe congenital heart disease and dependent upon a patent ductus for pulmonary perfusion. The administration of indomethacin to one infant was associated with a drop in arterial saturation which may have resulted from ductus narrowing (22). This observation is in accordance with the contraction of the ductus arteriosus ig utero obtained by giving indomethacin to dams prior to ----operative delivery (3). We have previously remarked on the possible consequences of intrauterine ductal closure on cardiovascular performana contracted ductal ce at birth (3). In these experiments, diameter in utero would retard significant blood flow through ________ this vessel and cause a changed hemodynamic situation with an increased load on the heart. ACKNOWLEDGEMENT We wish to acknowledge and thank Marianne Kjellberg and Inger Unnerstad for valuable technical assistance; and Ulla Kudryk for preparation of the manuscript and typing. REFERENCES 1.
2.
3.
594
of the ducF. and P. M. Olley. The response Coceani, tus arteriosus to prostaglandins, Canad. J. Physiol. Pharmacol. 51:220, 1973. Sharpe, G. L. and K. S. Larsson. Studies on closure of In vivo effect of prostathe ductus arteriosus. X. ------Prostaglandins (submitted). glandin, Sharpe, G. L., B. Thalme and K. S. Larsson. Studies on closure of the ductus arteriosus. XI. Ductal closure in utero by a prostaglandin synthetase inhibitor, -----_-Prostaglandins, 8:363, 1974.
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Ikeda, M., E. H. Rubinstein and R. R. Sonnenschein. Development of the 02 induced contractions in the ductus arteriosus of the guinea pig, Experientia 29:445, 1973. McMurphy, D. M., M. A. Heymann, A. M. Rudolph and K. L. Melmon. Developmental changes in constriction of the ductus arteriosus: Responses to oxygen and vasoactive agents in the isolated ductus arteriosus of the fetal lamb, Pediat. Res. 6:231, 1972. Boreus, L. O., T. Malmfors, D. M. McMurphy and L. Olson. .Demonstration of adrenergic receptor function and innervation in the ductus arteriosus of the human fetus, Acta physiol. stand. 77~316, 1969. McMurphy, D. M. and L. 0. Boreus. Studies on the pharmacology of the perfused human fetal ductus arteriosus, Amer. J. Obstet. Gynec. 109:937, 1971. KovalEl'k, V. The response of the isolated ductus arteriosus to oxygen and anoxia, J. Physiol. 169:185, 1963. Aronson, S., G. Gennser, C. H. Owman and N.-O. SjiSberg. Innervation and contractile response of the human ductus arteriosus, Europ. J. of Pharmacol. 11:178, 1970. Hbrnblad, P. Y. and K. S. Larsson. Studies on closure of the ductus arteriosus. I. Whole-body freezing as improvement of fixation procedures, Cardiologia 51:231, 1967. HBrnblad, P. Y. Studies on closure of the ductus arteriosus. III. Species differences in closure rate and morphology, Cardiologia 51:262, 1967. Hbrnblad, P. Y. Experimental studies on closure of the ductus arteriosus utilizing whole-body freezing, Acta Paediat. Stand. supp. 190, 1969. Traeger, A., H. Ndschel and J. Zaumseil. Zur Pharmakokinetik von Indomethacin bei Schwangeren, Kreissenden und deren Neugeborenen, Zbl. Gynlk. 95:635, 1973. Waltman, R., V. Tricomi and A. Palav. Aspirin and Effect on instillation/abortion time of Indomethacin. mid-trimester hypertonic saline induced abortion, Prostaglandins 3:47, 1973. Novy, M. J., M. J. Cook and L. Manaugh. Indomethacin block of normal onset of partuition in primates, Am. J. Obstet. Gynecol. 118:412, 1974. Prostaglandins during Pace-Asciak, C. and D. Miller. development. I. Age-dependent activity profiles of prostaglandin 15-hydroxydehydrogenase and 13, 14reductase in lung tissue from late prenatal, early postnatal and adult rats, Prostaglandins 4:351, 1973. of prostaglandin synthesis as a Vane, J. R. Inhibition mechanism of action for aspirin-like drugs, Nature New Biol. 231:232, 1971. bioFlower, R. J. Drugs which inhibit prostaglandin synthesis, Pharmacol. Rev. 26:33, 1974.
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19.
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Arcilla, R. A., 0. G. Thilenius and K. Ranniger. Congestive heart failure from suspected ductal closure 75:74, 1969. in utero J. of Pediat. __-_--__' cardiac failure associated Kohler, H. G. Intrauterine with premature closure of the ductus arteriosus. Arch. 1967. Dis. Child. 42:335, communication. Kohler, H. G. Personal Elliott, R. B., M. B. Starling and J. M. Neutze. Medical manipulation of the ductus arteriosus. Lancet I:140, 1975.
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