Changes in blood flow distribution during the perinatal period in fetal sheep and lambs Banting and Best Diabetes Centre and the Cerztre for Car8iovascular Research, Max Beld Research Centre, The Toronto Hospital and Department sf Pathology, fiiversity sf Toront8, Toronto, Ont., C a ~ a dM5G ~ 2C4
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Received July 13, 1992
BENDECK,M. P.,and LANGILLE, B. L. 1992. Changes in blood Wow distribution during the perinatal period in fetal sheep and lambs. Can. 9. Bhysiol. Pharmacol. 70: 1576- 1582. We have measured total blood flows and blood Wows per I00 g tissue to major tissues at 120 and 140 days gestation in fetal sheep and at 3 and 21 days of age in lambs (gestation period 144 i-2 days). Between 120 and 140 days gestation, flow per 100 g tissue increased by 74, 150, and 317% in the renal, intestinal, and hepatic arterial beds, but no further significant change in flow was observed at 3 or 21 days postpartum. Blood flows per 100 g to cerebral hemispheres and cerebellar tissues also increased dramatically during late gestation (142 and 121%, respectively), but declined sharply by 3 days post-
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partum (73 and 75 %, respectively). Brain blood Wows at 21 days postpartum remained substantially below late gestational levels. Adrenal blood flows per 100 g more than dc~ubledduring late gestation. fell by more tkan half at birth, and only pastially recovered by 21 days of age. Blood flows to carcass tissues did not change in late gestation, fell at birth, then partially recovered. Pre- and post-natal increases in brain blood flows were almost entirely attributable to increased perfusion rather than tissue growth, whereas large perinatal increases in flow to the diaphragm paralleled tissue growth. Tissue growth and increased perfusion per 100 g contributed almost equally to increased blood flows to kidneys postnavally, and to adrenal glands and the gastrointestinal tract prenatally. Key ~ w r d s blood : Wow perinatall birth, fetus, sheep.
BENDECK, M. P., et LANGILLE, B. L. 1992. Changes in blood flow distribution during the perinatal period in fetal sheep and lambs. Can. 9. Physiol. Pharmacol. 70 : 1576- 1582. Nous avons determine les d6bits sanguins totaux et les debits sanguins par 100 g de tissu aux tissus Hes plus importants, aux jours 120 et 140 de la gestation chez la brebis foetale. et B 1'8ge de 3 et 21 jours chez les agneaux (terme = 144 f 2 jours). Entre Bes jours 120 et 140 de la gestation, le dCbit par 100 g de tissu a augment6 de 74, 150 et 31'7% dans les Bits artkriels rknal, intestinal et hCpatique, mrris aucune autre variation significative n9a 6t6 observte aux jours 3 et 21 de postpartum. Les dkbits sanguins par 100 g aux h6rnisphkres cCrkbraux et aux tissus ckrkbelleux ont aussi augment6 considkrablement durant la dernikre phase de Ia gestation (142 et 121%, respectivernent), mais ont diminuC abmptement 3 jours aprks la parturition (73 et 75 74, respectivernent). Les debits sanguins cCrCbraux au jour 21 de postpartum sowt demeurCs sous les taux de la dernikre phase de la gestation. Les dkbits sanguins surrtnaux par 100 g de tissgls aussi snt plus que doublC durant la dernikre phase de la gestation, ils snt chute de plus de la moitik au nloment de la parturition et ne se sont que partiellement rktablis S 1'8ge de 21 jours. Les dkbits sanguins aux tissus de la carcasse n'ont pas variC durant la dernikre phase de la gestation, ils ont chute au nncmsent de la parturition, puis se sont partiellensent r6tablis. Les augmentations prC- et post-natales des dkbits sanguins ctrtbraux snt presque toutes kt6 atrribukes h une perfbasion accrue plut8t qu'au dkveloppement des tissus, tandis que les fortes augmentations perinatales du debit vers le diapkmgrne ont ktd paralBkles au dCveloppement tissulaire. Le dkveloppement tissulaire et la perfusion accrue par 180 g ona contribuk presque Cgalement 5 l'augmentation des debits sanguins vers les reins en pCriode postnatale et vers les glandes surrCnales et le tractus gastrointestinal en phase prknatale. Mots ci6.r dkbit sanguin, pkrinatal, naissance, foetus, brebis. [Traduit par la rkdaction]
Introduction There are large and abrupt changes in cardiovascular function during the perinatal period. Several studies have reported a dramatic redistribution of peripheral blsod flows at parturition as a result of increased arterial oxygen content, closure of fetal vascular shunts, and alterations in the metabolic function of various organs. Heymann et QB. (1981) reported large increases in blood flow to the lungs, heart, kidneys, and gastrointestinal tract and decreases in flow to cerebral, adrenal, skin, muscle, and bone tissues between late gestation and early postnatal life, although the exact ages at which measurements were made were not given. Wosenkerg et a / . (1984) reported similar findings, and they also found large increases in blood flows to 'Present address: Department of Pathology, SJ-60, University of Washington, Seattle, Wash. 98 195, U.S.A. 'Correspondence mlay be sent to the author at the following address: Max Bell Research Centre, The Toronto Hospital, 200 Elizabeth St., Toronto, Ont., Canada M5G 2C4. Pr~ntcdin Candda , Irnpr~rnCau Canada
liver. spleen, and diaphragm between 130 days gestation and 3 - 10 days postpartum. By contrast, Richardson et a&.(1989) did not observe increases in renal or gastrointestinal blood WOWS immediately after birth. Instead, these blood flows at 2 or 24 h postpartum were not different from prenatal values ( 139- 142 days gestation). These previous studies measured changes in blood flow distribution at one time point before parturition and at one or two time points after. Consequently, it was not possible to compare changes that occurred at parturition with those occurring during adjacent periods. In the current study, we measured tissue blood flows in sheep at four time points, which encompassed 5 -6 weeks surrounding birth. As in previous studies, radioactive microsphere techniques were used to measure blood flow per unit tissue weight; however, we also determined total tissue blsod flows to enable us to distinguish between increases in flow linked to tissue growth versus those related to changing metabolic demands s f the tissues. Both total and weight-specific blood Wows to many tissues
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BENDECK AND LANGlLLE
increased during late gestation in the fetal lamb. At birth, there were large decreases in both total and weight-specific flows to the cerebral hemispheres and the cerebellum, and there were decreases in tissue blood flow per 100 g in the carcass, skin, bone, and adrend glands. No tissues exhibited increases in blosd flow per 100 g between 140 days gestation and either 3 or % Idays of age. Instead, our data suggest that increased perfusion (per 100 g) of gut and renal tissues between 130 days gestation and 3 - 10 days postpaaum (Rosenberg eb a / . 1984) is largely attributable to late gestational changes rather than the acquisition of postnatal function in these organs.
Methods SurgicaI iastrurnenfatiora Throughout these studies, all animals were cared for in accord with the principles of the Guide to the Care and Use of Experimental AnimL produced by the Canadian Council on Animal Care. Fetal sheep were studied at 120 and 140 days gestation (gestation period = 14-4 f 2 days), and newborn lambs were studied at 3 and 21 days postpartum. For fetal studies, pregnant ewes at 116 or 136 days gestation were anesthetized with thiopental sodium (I g. i.v.), inmbated, and artificially ventilated. Anesthesia was maintained with 1-2% halothane in oxygen, and lactated Ringer's (500 m%/h) was given intravenously. The uterus was exposed through a midline abdominal incision. The following studies were then carried out on the fetus. The fetal hind limbs were withdrawn through a uterine incision, and the uterine wall was sealed to the fetus with Babcmk clamps to prevent loss of amniotic fluid. Catheters were inserted into the abdominal aorta via the femoral artery, and into the inferior vem cava via the femoral vein (V4 vinyl catheters, Bolab, Lake Havasu City, Ariz.). A catheter was mounted on the skin over the leg to record amniotic pressure (V 11 vinyl catheter, Bolab , Lake Mavasu City9Ariz.). The hind limbs were reinserted into the uterus, the head was withdrawn, and the left common carotid artery was catheterized. The fetus was returned to the uterus and the uterine incision closed. The catheters were exteriorized through a small incision in the flank sf the ewe, and the abdominal incision of the ewe was closed. Catheters were stored in clean plastic bags tucked under No. 8 tubular elastic net bandage (Medical Mart, Mississauga, Ont.) wrapped around the ewe's abdomen. Postoperative analgesia was provided using 5 mg Numorphen suppositories (Du Bont Chemicals Inc., Scarborough, Ont.). The ewes were housed in metabolic cages, and catheters were flushed twice daily with sterile heparinized saline (20 U/mL) to maimtain patency. Amniotic pressure was recorded continuously on a Grass model 78 polygraph (Grass Instruments, Quincy , Mass.), by coupling catheters to Statham P23 pressure transducers (Gould Inc., Oxnard, Calif.). Radioactive microsphere determinations of blood flow were performed at 120 or 140 days gestation (see below). Surgery was also performed on ewes at 140- 144 days gestation as described above. A catheter was advanced into the left ventricle of the fetus via the left common carotid artery. Position of the catheter tip was determined by coupling the catheter to a pressure transducer and chart recorder and noting when ventricular wave forms were observed. Another catheter was placed in the abdominal aorta via the femoral artery. Arterial pressure was measured daily as described above. When amniotic pressures indicated the onset of labour, catheters were knotted and cut close to their point of exit from the ewe. The ewes delivered lambs vaginally with staff in attendance. Catheters readily passed through the maternal exit site and were delivered with the lambs. They were recoupled to stopcocks. then packaged in the pockets of nylon jackets worn by the lambs. Newborn lambs were fed for at least 1 day by the ewe, to ensure they received coIostmm. Subsequently, they were bottle fed 3 times per day with a commercial ewes' milk replacement. Microsphere measurements of blood flow were performed 3 days after birth (see below).
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Surgery was also performed on 14-day-old lambs. The lambs were anesthetized with 1 - 1.5 % halothane in 0,that was delivered via a face mask. Under sterile conditions, a catheter was inserted retrogradely into the left carotid artery and advanced into the left ventricle, and a second catheter was advanced into the abdominal aorta via the femoral artery. Microsphere measurements of blood flow were performed at 21 days s f age (see below). For most preparations, arterial blosd Pao,, Paco,, and pH were measured every other day from a 1-mL sample of blood using a Corning model 170 pHIblood gas analyzer (Corning Medical and Scientific, Medfield, Mass.). kfeasurernents of blood flow, using mdiocrctdve microspheres Procedures for microsphere determinations of blood flow were adapted from Heyrnann eb al. (197'7). Different injection and withdrawal sites were chosen for fetuses and lambs because of the unique arrangement of the fetal circulation. In the fetus, spheres injected into the inferior vena cava pass via two routes to the systemic circulation (right heart and ductus, or foramen ovale and left heart) to deliver different concentrations of microspheres to the upper and lower body. For this reason, reference samples were withdrawn simultaneously from the fetal carotid and femoral arteries. Postpartum, microspheres in-jected into the left ventricle are delivered in equal concentrations to all systemic tissues, so a single (femoral) reference sample was employed. Hn fetuses, the femoral and carotid arterial catheters were coupled to a Harvard m d e l 8-945 infusion -withdrawal pump (Harvard Apparatus, South Nantick, Mass.). Withdrawal of blood was inif ated at a rate of 2.5 mL/rnin9 then 0.5 X lo6 microspheres/kg body weight (estimated) were injected immediately via the femoral vein catheter. Microspheres were 15 pm in diameter and were labelled with 4 6 S (3M, ~ New Brighton, Minn.). Blood withdrawal was continued for a total of 2.5 min. Additional samples of 0.20 mL of blood withdrawn from each catheter yielded only background radioactivity, confirming that an adequate withdrawal duration had been employed. Thirty minutes after microsphere injection, the ewe and fetus were euthanized by injecting 20 mL of T-61 (2W mglml N-1-2-rn-methoxyphenyl-2-ethylbutyl-(1)1-2-hydroxybutyramide 50 m g / d 4,4 '-methylene-bis(cyclohexylt~e~y1ammonium iodide), and 5 m g / d tetracaine HCl; Homhst Canada Inc.. MontrCal, Que.) into the jugular vein of the ewe. Abdominal and uterine incisions were opened, the umbilical cord was tied, and the fetus was removed. All cotyledons were carefully excised from the utems; they were cut from the fetal side of the utems to ensure that all fetal membranes were included in the sample. Total radioactivity of cotyledons was used to determine placental blood flow. Hn lambs, procedures were as for fetuses except that microspheres were injected into the left ventricle and a single reference sample was withdrawn from the femoral arterial catheter. Thirty minutes after sphere injection, the lamb was sacrificed by injection of 1 mL of T-61 into the femoral artery.
Blood j b w and cardiac output determinations Tissues were carefully excised, weighed, minced, and dried in an oven at 60°C. The tissues were ground in a blender, then poured into plastic test tubes to be counted using a y-counter (Cornpugamma, Pharmacia LKB, Cambridge, England). Blood flows to tissues were determined using the formula (Heyrnann et ab. 1977)
where Q, is blood flow to the tissue sample. Crrepresents counts per minute recorded from the reference sample, Q, is the withdrawal rate of this reference sample in millilitres per minute, and Cs represents the counts per minute recorded from the tissue sample. Blood flows per 100 g tissue to the right and left kidneys, right and left cerebral hemispheres, and to skin on the right and left side of the head were compared to ensure adequate mixing of the spheres. Adequate mixing was confirmed since variability between left and right side blood flows withln animals was much less than variability among
CAN. J. PHYSHBL. PHARMACOL. VOL. 70, 1992
TABLEI . Perinatal blood gases and CO
Pao, (rnmHg) Paco, (mmHg) PH CVO (mE/(min kg)) C 0 (rnL/(min = kg))
20.8+0.6(3) 44.8f 1.4 (3) 7.42f 0.01 (3) 363 f 46 (5)
14.5+1.7(6) 49.4k0.9 (4) 7.39f 0.82 (6) 335 f37 (5)
69.9+3.1(4) 42.0f 3.5 (4) 7.41 k0.804 (4) 412181 ( 5 ) 206 f57 (5)
87.7k0.7 (2) 34.7k0.7 (2) 7.48f0.00 (2) 414116 (5) 207 1 1 (5)
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NOTE: Values are mean f SEM. Numbers in parentheses are sample sizes. dg, days gestation; do. days postpartum.
TABLE2. Perinatal organ blood flows per 100 g tissue wet weight Organ
120 dg
140 dg
3 do
21 do
Cerebral hemisphere Cerebellum Intestine Kidney Liver (hepatic artery) Adrenal Spleen Diaphragm Perirenal fat Skeletal muscle Skin Bone Subdiaphragmatic carcass NOTE: Values are mean k SEM. rz = 6 for 140 days gestation. n = 5 all other data, except n = 4 for skin and cerebral hemisphere at 3 days. dg, days gestation; do, days postpartum. "Significantly different ( p < 0.05) from value measured at 140 days gestation.
animals for all three tissues. Specifically, we found that the standard error between left and right side samples was much less than the among-animal standard error for either sample. This was true for both skin and kidney blood Wows at all ages. There were no obvious outliers. Total organ blood flow was measured, and blood Wow per 100 g tissue wet weight was calculated for several organs, including the placenta (all cotyledons), right and left cerebral hemispheres, cerebellum, kidneys, adrenals, diaphragm, liver and gallbladder, the subdiaphragmatic carcass, and the portion of the intestine supplied by the superior mesenteric artery (see below). In addition, samples of skin from the chest wall, hind-limb muscle and bone, and perirenal fat were excised and weighed, and blood flows per 100 g tissue wet weight were determined. Subdiaphragmatic carcass flow was calculated by counting the tissues of the subdiaphragmatic amnk and abdominal wall, and doubling right hind limb flow measurements. The liver and gallbladder were excised and counted together for determination of hepatic arterial blood Wow. We assume that the diaphragm is supplied entirely via the celiac artery in fetal and neonatal lambs; thus, blood flow was calculated by comparison with the femoral artery reference sample. This measurement is subject to a minor error in the fetal lambs, since a small proportion s f diaphragmatic flow is supplied by the mammary artery, which branches from the aorta upstream of the ductus arteriosus and carries output from the left ventricle. In newborn lambs, cardiac outputs (CO) were calculated using the formula (Heymann ef al. 1977)
where Q,represents the withdrawal rate of the femoral artery reference sample, Ct is total counts per minute injected, and Cr is counts per minute recorded from the femoral artery reference sample. In fetuses, combined ventricular output (CVO) was estimated using the equation
where Qc represents withdrawal rate of the carotid artery reference sample, Ct is total counts per minute injected, Q,is withdrawal rate of the femoral artery reference sample, Cf is counts per minute in the femoral artery reference sample, and Cc is counts per minute in the carotid artery reference sample. This estimate is derived from equation 1, based on the assumption that lower body blood flow is 3 X upper body flow in fetal sheep (Rudolph 1985). It also assumes that counts per minute in blood delivered to the lungs and the Bower body are equal. This latter assumption introduces a small error as a result of the minor contribution of left ventricular output to lower body blood flows.
Data analysis Sample sizes for b l o d Wow measurements were pm = 5 per group for 120 day gestation fetuses and 3- and 21-day-old lambs, and az = 6 for 140 day gestation fetuses. Counts were not recovered for skin and cerebral hemispheres for one 3-day-old lamb, so n = 4 for these data. Dunnett's tests were used 6 0 determine significant differences in blood flow and pressure at different ages. Our primary goals were to test for significant changes in blood flow and pressure during late gestation and to measure changes associated with birth as well as later during the postnatal period. Consequently, blood flows and pressures at 120 days gestation and at 3 and 21 days postpartum were compared with flows and pressures near full term (140 days gestation).
Results B&oodgases and cardiac output Table 1 displays data on blood gases, CO, and estimated CVO in the perinetal period. fio2, Paco2, and pH (Table 1) were in accord with previous measurements from fetal and neonatal blood samples (Richardson et al. 1989). Estimated CVO did not change significantly during late gestation (120148 days) . CVO was slightly, but not significantly, above fetal levels at 3 days and was unchanged at 21 days. We observed no change in CO (left ventricular output) per kilogram body
BENDECK AND LANSILLE
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(A)
Cerebral Hemispheres
Kidneys
Cerebellum 6-%
t
28
1
FIG. 1. Total tissue blood Wow (rnklmin), measured at 120 and 140 days gestation and 3 and 21 days postpartum in (A) right (a) and left ( 3 )cerebral hemispheres and (B) cerebellum. Values are mean k SEM . n = 4 - 6 per group. "Significantly different from value at 140 days gestation, p < 0.05. Vertical broken rule indicates mean gestational age at parturition.
weight between 3 (206 f 57 mL/(min . kg)) and 21 days (207 f 11 mL/(min * kg)) postpartum. Placental blood flow (not shown) was 657 38 rnL/rnin at 120 days gestation and was not significantly different (557 42 mL/min) at 148 days gestation.
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'irissue blood jbws
Total tissue blood flows are shown in Figs. 1-3. As in many tissues, brain blood flows increased in late gestation (Fig. 2). Between 120 and 140 days gestation, total right and left cerebral hemisphere blood flow increased by 158% and total cerebellar flow increased by 204 % . At birth, cerebral and cerebellar blood flows decreased dramatically (by 70% between 140 days gestation and 3 days postpartum). Blood flows to brain tissues then increased, but remained below fetal levels at 21 days postpartum. Cerebral hemisphere flows increased by 122%, from 16.1 &- 2.6 mL/rmin at 3 days post2.3 mL/min at 21 days postpartum, and partum to 35.8 cerebellar flow increased by 67%, from 4.68 f 0.91 to 7.8 1 +_ 0.57 mL/min between 3 and 2 1 days postpartum. The same trends were observed for cerebral and cerebellar blood
+
FIG. 2. Total tissue blood flow (mklmin), measured at 128 and 140 days gestation and 3 and 21 days postpartum in (A) kidneys and (B) adrenals. Values are mean 9 SEM. n = 5 -6 per group. "Significantly different from value at 148 days gestation, g < 0.05. Vertical broken rule indicates mean gestational age at parturition.
flows per 180 g tissue (Table 2), primarily because brain weights did not change postpartum. No differences were seen in perfusion of left and right hemispheres at any time, which probably indicates that collateral supply to the left hemisphere totally compensated for catheterization of the left carotid artery in fetuses. Total renal blood flows nearly doubled in late gestation (Fig. 2A), from 27.2 3.1 mL/min at 128 days gestation to 5 1.0 3.6 mL/min at 140 days gestation, largely as a result of increased perhsion per 180 g weight (Table 2). No change in renal perhsion accompanied parturition, but total blood flows again rose dramatically, by 3-fold, by 21 days postpartum. However, half of this increased total blood flow was due to increased renal tissue weight and half was a result of increased relative tissue perfusion, since there was a 3-fold increase in total blood flow but only a 1.5-fold increase in blood flow per 180 g tissue weight (Table 2). A 5-fold increase in adrenal blood flow, from 0.690 f 8.080 mL/min at 120 days gestation to 3.49 f 0.25 mL/min at 148 days gestation, was again due to approximately equal increases in tissue weight and relative tissue perhsion. There was no further significant change in total blood flow at the
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CAN. J . PHYSIOL. PHARMACOL. VOL. 70, 1992
(A)
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intestine
Diaphragm
Subdiaphragmatic Carcass
Hepatic Artery T
FIG.3. Total tissue blood flow (mE/min), measured at 120 and 140 days gestation and 3 and 21 days postpartum in (A) intestines and (B) hepatic arterial circulation. Values are mean -t SEM. n = 5 or 6 per group. "Significantly different fron-n value at 240 days gestation, p < 0.05. Vertical broken rule indicates mean gestational age at parturition.
postpartum times we assessed. although considerable variability between animals was observed (Fig. 2B). Substantial increases in blood flows to the gastrointestinal tract were concentrated in the prenatal period. Intestinal blood flows increased by 4-fold in late gestation (Fig. 3A). Again, tissue growth and increased relative tissue perfusion contributed equally. No changes occurred in the immediate perinatal period, and after birth, a doubling of mean intestinal blood Wows was not statistically significant, as a result of high variability between animals. Hepatic arterial blood Rows to liver and gall bladder also increased 4-fold in late gestation, from 4.10 1.26 rnl/rnin at 120 days gestation to 17.1 f 4.8 rnl/rnin at 140 days gestation (Fig. 3B). but in this case, the increase was due entirely to a 4-fold increase in perfusion per 1100 g tissue (Table 2). No significant increases were recorded at times after 140 days. Splenic blosd Rows showed no significant changes in the perinatal period (Table 2). Diaphragmatic blood flows were surprisingly consistent between fetal preparations, given that no attempt was made to define whether flow measurements coincided with episodes of
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FIG.4. Total tissue blood flow (mL/min), measured at 120 and 140 days gestation and 3 and 21 days postpartum in (A) diaphragm and (B) subdiaphragmatic carcass. Values are mean f SEM. n = 5 or 6 per group. "Signiiicantly different from value at 140 days gestation, g < 0.05. Vertical broken rule indicates mean gestational age at parturition.
fetal breathing movements. Total diaphragmatic blood Wows doubled in %ategestation, rising from 1.33 k 0.37 rnl/rnin at 728 days to 2.69 f 0.30 mL/min at 140 days, and doubled again between 3 and 2 1 days postpartum (from 3.75 f 0.63 to 8.44 a 1.73 rnL/min) (Fig. 4A), These changes were due to increases in tissue mass, since there were no changes in perfusion per 100 g tissue. Strikingly, no changes in perfusion of diaphragmatic tissues were seen between 148 days gestation and 3 days postpartum, despite the initiation of lung gas exchange (Fig. 4A). Total blood flow to subdiaphragmatic carcass tissues showed relatively modest changes in the perinatal period (Fig. 4B) and only those changes occurring between 120 and 140 days gestation proved significant, although high data variability may have masked increases after birth. Mean flows changed by nearly 2-fold over this time. The only significant change in carcass blood flows per 180 g tissue was a 42 % fall between 140 days gestation and 3 days postpartum (Table 2). Blood flow to bone per 180 g was halved in late gestation (Table 2). These flows showed another dramatic decrease, by 84% after birth, before recovering slightly between 3 and 21
BEWDECK AND EANGIEEE
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days postpartum. Relative perhsion of skin did not change in late gestation, but fell by 88% postparturn before rising to about half of fetal levels by 29 days postpartum. Although mean blood flow per 188 g to perinatal fat doubled between 120 and 140 days gestation, then decreased dramatically by 21 days postpartum, these changes were not significant as a result s f variability between animals in the immediate perinatal period.
Discussion There are dramatic changes in cardiovascular function during the perinatal period. Changes directly associated with birth include closure of fetal vascular shunts (ductus arteriosus, foramen ovale, ductus venosus), increase in systemic and decrease in pulmonary arterial pressure, greatly increased pulmonary blood flow, loss of placental perfbsion, and increased left ventricular output (Heymann st a / . 1981). Our measurements of CVO in fetal sheep and CO in 2 1-day-old lambs were in accord with those of others (Lister et ak. 1999; Klopfenstein and Rudolph 1978; Rosenberg et al. 1984). However, some previous studies have reported large, transient increases in left ventricular output in the lamb from 150 before birth to -480 ml/(min kg) by 0-3 days after birth (Lister et ak. 1979; Klopfenstein and Rudolph 1978). Our measurements of left ventricular output after birth are not consistent with these data. We measured outputs of 280 ml/(min . kg) at 3 days postpartum. These outflows are undsubtedly above fetal levels, since the left ventricle delivers less than half of the 358 ml/(min - kg) of CVO in utero (Rudolph 1985); nonetheBess, they are well below the values reported in the two previous studies. The reason for this discrepancy is unclear. Recent studies with human subjects indicate that left ventricular output is twice fetal levels at 1 h after birth, but output declines to 44% above fetal values by 24 h and then doesn't change over the subsequent 3 days (Agata et a!. 1991). These data are consistent with our finding of moderately elevated left ventricular output at 3 days. In addition to these changes in central circulatory function, there were large changes in peripheral tissue blood flows throughout the perinatal period. However, one of our most striking findings was that no tissues exhibited increased perfusisn per unit tissue mass between late gestation (140 days) and early postpartum life ((3 or 21 days). Previous investigators have reported increases in brain, hepatic arterial, intestinal, renal, splenic, and diaphragmatic blood flows per 100 g tissue when measurements made at 3 - 10 days postpartum were compared with a single prepartum value (Rosenberg ef a / . 1984); however, our data indicate that many of these increases occur during late gestation. This observation has important implications in that it suggests that late gestational growth and maturation of tissues are major determinants of changing tissue perfusion. Late gestational increases in flow to the diaphragm, skin, and muscle largely paralleled tissue growth, and perfusion per 100 g did not change significantly; however, increases in adrenal, intestinal, brain, and renal blood flows exceeded those attributable to growth, and flows per 108 g approximately doubled. Intestinal blood flows may increase in late gestation as a result of the metabolic demands imposed by maximal proliferation of lymphoid cells in Peyer9spatches at this time (Reynolds and Morris 1983), whereas increased brain blood flow probably reflects high levels of cerebral angiogenesis despite slowed brain growth in the perinatal
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*
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period (Bar 1983). Bone blood flows were halved between 128 and 140 days gestation, but the reason for this decrease is unclear. These results underscore the importance of both growth and altered tissue metabolism in influencing late gestational blood flow changes. Our failure to detect increased perfusion at birth to any of the tissues that were examined reflects, in part, increased blood oxygen content after parturition. Thus, Pao2 increased from about 20 to almost 90 mmHg (I mmHg = 133.3 Pa) over the ages we examined, a finding consistent with previous reports (Richardson et a/. 1989; Rosenberg et al. 1984). This increased Pao2 reduces perfbsion demands, at least in terms s f O2 requirements, and this undoubtedly accounts for the postpartum fall in blood flow to skeletal muscle, brain, and adrenal glands. This interpretation is consistent with the data of Iwamoto et al. (1989), who measured decreases in brain, adrenal, carcass, and heart blsod flows per tissue weight after ventilating the lungs of fetuses in utero. Also, Peeters et a&. (1979) increased fetal blood oxygen content by administering hyperbaric oxygen to pregnant ewes and measured decreased brain, heart, and adrenal flows per tissue weight. For some tissues whose blood supply is not determined primarily by gasexchange requirements, like the kidneys, no change in perfbsion occurred at parturition. Other studies have reported no change in renal or gastrointestinal blood flow per weight between birth and 3 days postpartum (Richardson et al. 1989; Aperia et al. 1977; Nakamura er al. 1989). In contrast, a marked fall in skin blood flow at parturition was probably due to a combination of increased O2 levels and a thermoregulatory response to heat loss in the extrauterine environment. Changes in blood flow distribution immediately surrounding birth are also undoubtedly influenced by dramatic changes in plasma concentrations of catecholamines, angiotensin 11, arginine vasopressin, and prostaglandins E and F (Heymann et al. 1981; Mott 1975; Eliot et a / . 1981; Alexander et al. 1972). Thus, for example, Alexander st al. (1972) have demonstrated the capacity of catecholamines to markedly redistribute blsod flows in newborn lambs. Blood flows to many tissues increased between 3 and 21 days postpartum, although no flows attained values that exceeded perfbsion at 148 days gestation. In summary, we have measured marked increases in total blood flow and perfusion per unit weight in many tissue beds during late gestation. These findings underscore the important influence of late gestational growth and maturation of tissues on blood flow distribution. Large decreases in cerebral, cerebellar, adrenal, skin, bone, and carcass perhsion occurred between 140 days gestation and 3 days postpartum, but there were no significant changes in flow to other tissues at birth. Furthermore, there were no increases in blood flow per weight in any tissue between late gestation and 3 weeks of age. Previous inferences that increases in gut and kidney blood flows between 130 days gestation and 3- 10 days postpartum are attributable to the acquisition of postnatal function in these organs (Rosenberg et dl. 1984) are not supported by our data, which indicate that increased perfusion of these organs occurs mainly during late gestation and is not directly associated with birth.
Acknowledgements The authors acknowledge the technical assistance of Doreen LeBlanc. This research was supported by the Medical Research Council of Canada (MRC). M . P. Bendeck is an awardee of an MRC Studentship. B. L. Langille is a Career Investigator of the Heart and Stroke Foundation of Ontario.
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CAN. J . PHYSIOL. PHARMACOL. VOL. 70, 1992
Agata, Y.. Hiraishi, S., Oguchi, K., Misawa, H.,Horiguchi, Y., Fujino, N., Yashirs, K., and Shimada, N. 1991. Changes in left ventricular output from fetal to early neomatal life. J. Pediatr. (St. Louis), 119: 44 1 -445. Alexander, G., Bell, A. W., and Setchell, B. P. 1972. Regional distribution of cardiac output in young lambs: effect of cold exposure and treatment with catecholamines. J. Physiol. (London), 228: 51 1-528. Aperia, A., Broberger, O . , Merin, P., and Joelsson, 1. 1977. Renal hemodynamics in the perimtal period. Acta Physiol. Scand. 99: 261 -249. Bar, T. 1983. Patterns of vascularization in the developing cerebral cortex. In Development of the vascular system. Pitman, London. pp. 20-36. Eliot, R. J., Klein, A. H o gGlatz, T. H., Nathanielsz, P. W., and Fisher, D. A. 1981. Plasnaa norepinephrine, epinephrine, and dopamine concentrations in maternal and fetal sheep during spontaneous parturition and in premature sheep during cortisol-induced parturition. Endocrinology (Baltimore), 108: 1678 - 1682. Heymann, M. A., Payne, B. B., Woffmn, J. I. E., and Rudolph, A. M. 1977. Blood flow measurements with radio-nuclide-labeled particles. Prog. Cardiovasc. Dis. 20: 55-77. Heymann, M. A., Hwamoto, H. S., and Rudolph, A. M. 1981. Factors affecting changes in the neonatal systemic circulation. Annu. Rev. Physiol. 43: 371 -383. Iwamoto, H. S., Teitel, D., and Rudolph, A. M. 1987. Effects of birth-related events on blood Wow distribution. Pediatr. Res. 22: 634-640.
Klopfenstein, H. S., and Rudolph, A. M. 1978. Postnatal changes in the circulation and responses to volume loading in sheep. Circ. Res. 42: 839-845. Lister, G., Walter, T. K., Versmold, H. T., Dallman, P. R., and Rudolph, A. M. 1979. Oxygen delivery in lambs: cardiovascular and hematologic development. Am. J . Physiol. 237: H668 - H675. Mott, J. C. 1975. TRe place of the renin - angiotensin system before and after birth. Br. Med. Bull. 31: 44-50. Nakamura, K. T., Matherne, G. P., McWeeney, 0 . J., Smith, B. S., and Wobillard, J. E. 1987. Renal hennodynamics and functional changes during the transition from fetal to newborn life in sheep. Pediatr. Res. 21: 229-234. Peeters, L. L. H., Sheldon, R. E., Jones, M. B., Makowski, E. L., and Meschia, G. 1979, Blood Wow to fetal organs as a function of arterial oxygen content. Am. J. Obstet. Gynecol. 135: 637-646. Reynolds, J. B., and Morris, B. 1983. The evolution and involution of Peyer's patches in fetal and postnatal sheep. Eur. J. Immunol. 13: 627-635. Richardson, B. R., Carrnichael, L., Homan, J. Tanswell, K., and Webster, A. C. 1989. Regional b l o ~ dflow change in the lamb during the perinatal period. Am. J. Obstet. Gynecol. 160: 919 -925. Wosenberg, A. A., Koehler, 8 . C., and Jones, M. D., Jr. 1984. Bistribution of cardiac output in fetal and neonatal lambs with acute respiratory acidosis. Pediatr. Res. 18: 73 1 -735. Rudolph, A. M. 1985. Distribution and regulation of blood Wow in the fetal and neonatal lamb. Circ. Res. 57: 8 11- 82 1.
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Received July 13, 1992
BENDECK,M. P.,and LANGILLE, B. L. 1992. Changes in blood Wow distribution during the perinatal period in fetal sheep and lambs. Can. 9. Bhysiol. Pharmacol. 70: 1576- 1582. We have measured total blood flows and blood Wows per I00 g tissue to major tissues at 120 and 140 days gestation in fetal sheep and at 3 and 21 days of age in lambs (gestation period 144 i-2 days). Between 120 and 140 days gestation, flow per 100 g tissue increased by 74, 150, and 317% in the renal, intestinal, and hepatic arterial beds, but no further significant change in flow was observed at 3 or 21 days postpartum. Blood flows per 100 g to cerebral hemispheres and cerebellar tissues also increased dramatically during late gestation (142 and 121%, respectively), but declined sharply by 3 days post-
-
partum (73 and 75 %, respectively). Brain blood Wows at 21 days postpartum remained substantially below late gestational levels. Adrenal blood flows per 100 g more than dc~ubledduring late gestation. fell by more tkan half at birth, and only pastially recovered by 21 days of age. Blood flows to carcass tissues did not change in late gestation, fell at birth, then partially recovered. Pre- and post-natal increases in brain blood flows were almost entirely attributable to increased perfusion rather than tissue growth, whereas large perinatal increases in flow to the diaphragm paralleled tissue growth. Tissue growth and increased perfusion per 100 g contributed almost equally to increased blood flows to kidneys postnavally, and to adrenal glands and the gastrointestinal tract prenatally. Key ~ w r d s blood : Wow perinatall birth, fetus, sheep.
BENDECK, M. P., et LANGILLE, B. L. 1992. Changes in blood flow distribution during the perinatal period in fetal sheep and lambs. Can. 9. Physiol. Pharmacol. 70 : 1576- 1582. Nous avons determine les d6bits sanguins totaux et les debits sanguins par 100 g de tissu aux tissus Hes plus importants, aux jours 120 et 140 de la gestation chez la brebis foetale. et B 1'8ge de 3 et 21 jours chez les agneaux (terme = 144 f 2 jours). Entre Bes jours 120 et 140 de la gestation, le dCbit par 100 g de tissu a augment6 de 74, 150 et 31'7% dans les Bits artkriels rknal, intestinal et hCpatique, mrris aucune autre variation significative n9a 6t6 observte aux jours 3 et 21 de postpartum. Les dkbits sanguins par 100 g aux h6rnisphkres cCrkbraux et aux tissus ckrkbelleux ont aussi augment6 considkrablement durant la dernikre phase de Ia gestation (142 et 121%, respectivernent), mais ont diminuC abmptement 3 jours aprks la parturition (73 et 75 74, respectivernent). Les debits sanguins cCrCbraux au jour 21 de postpartum sowt demeurCs sous les taux de la dernikre phase de la gestation. Les dkbits sanguins surrtnaux par 100 g de tissgls aussi snt plus que doublC durant la dernikre phase de la gestation, ils snt chute de plus de la moitik au nloment de la parturition et ne se sont que partiellement rktablis S 1'8ge de 21 jours. Les dkbits sanguins aux tissus de la carcasse n'ont pas variC durant la dernikre phase de la gestation, ils ont chute au nncmsent de la parturition, puis se sont partiellensent r6tablis. Les augmentations prC- et post-natales des dkbits sanguins ctrtbraux snt presque toutes kt6 atrribukes h une perfbasion accrue plut8t qu'au dkveloppement des tissus, tandis que les fortes augmentations perinatales du debit vers le diapkmgrne ont ktd paralBkles au dCveloppement tissulaire. Le dkveloppement tissulaire et la perfusion accrue par 180 g ona contribuk presque Cgalement 5 l'augmentation des debits sanguins vers les reins en pCriode postnatale et vers les glandes surrCnales et le tractus gastrointestinal en phase prknatale. Mots ci6.r dkbit sanguin, pkrinatal, naissance, foetus, brebis. [Traduit par la rkdaction]
Introduction There are large and abrupt changes in cardiovascular function during the perinatal period. Several studies have reported a dramatic redistribution of peripheral blsod flows at parturition as a result of increased arterial oxygen content, closure of fetal vascular shunts, and alterations in the metabolic function of various organs. Heymann et QB. (1981) reported large increases in blood flow to the lungs, heart, kidneys, and gastrointestinal tract and decreases in flow to cerebral, adrenal, skin, muscle, and bone tissues between late gestation and early postnatal life, although the exact ages at which measurements were made were not given. Wosenkerg et a / . (1984) reported similar findings, and they also found large increases in blood flows to 'Present address: Department of Pathology, SJ-60, University of Washington, Seattle, Wash. 98 195, U.S.A. 'Correspondence mlay be sent to the author at the following address: Max Bell Research Centre, The Toronto Hospital, 200 Elizabeth St., Toronto, Ont., Canada M5G 2C4. Pr~ntcdin Candda , Irnpr~rnCau Canada
liver. spleen, and diaphragm between 130 days gestation and 3 - 10 days postpartum. By contrast, Richardson et a&.(1989) did not observe increases in renal or gastrointestinal blood WOWS immediately after birth. Instead, these blood flows at 2 or 24 h postpartum were not different from prenatal values ( 139- 142 days gestation). These previous studies measured changes in blood flow distribution at one time point before parturition and at one or two time points after. Consequently, it was not possible to compare changes that occurred at parturition with those occurring during adjacent periods. In the current study, we measured tissue blood flows in sheep at four time points, which encompassed 5 -6 weeks surrounding birth. As in previous studies, radioactive microsphere techniques were used to measure blood flow per unit tissue weight; however, we also determined total tissue blsod flows to enable us to distinguish between increases in flow linked to tissue growth versus those related to changing metabolic demands s f the tissues. Both total and weight-specific blood Wows to many tissues
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BENDECK AND LANGlLLE
increased during late gestation in the fetal lamb. At birth, there were large decreases in both total and weight-specific flows to the cerebral hemispheres and the cerebellum, and there were decreases in tissue blood flow per 100 g in the carcass, skin, bone, and adrend glands. No tissues exhibited increases in blosd flow per 100 g between 140 days gestation and either 3 or % Idays of age. Instead, our data suggest that increased perfusion (per 100 g) of gut and renal tissues between 130 days gestation and 3 - 10 days postpaaum (Rosenberg eb a / . 1984) is largely attributable to late gestational changes rather than the acquisition of postnatal function in these organs.
Methods SurgicaI iastrurnenfatiora Throughout these studies, all animals were cared for in accord with the principles of the Guide to the Care and Use of Experimental AnimL produced by the Canadian Council on Animal Care. Fetal sheep were studied at 120 and 140 days gestation (gestation period = 14-4 f 2 days), and newborn lambs were studied at 3 and 21 days postpartum. For fetal studies, pregnant ewes at 116 or 136 days gestation were anesthetized with thiopental sodium (I g. i.v.), inmbated, and artificially ventilated. Anesthesia was maintained with 1-2% halothane in oxygen, and lactated Ringer's (500 m%/h) was given intravenously. The uterus was exposed through a midline abdominal incision. The following studies were then carried out on the fetus. The fetal hind limbs were withdrawn through a uterine incision, and the uterine wall was sealed to the fetus with Babcmk clamps to prevent loss of amniotic fluid. Catheters were inserted into the abdominal aorta via the femoral artery, and into the inferior vem cava via the femoral vein (V4 vinyl catheters, Bolab, Lake Havasu City, Ariz.). A catheter was mounted on the skin over the leg to record amniotic pressure (V 11 vinyl catheter, Bolab , Lake Mavasu City9Ariz.). The hind limbs were reinserted into the uterus, the head was withdrawn, and the left common carotid artery was catheterized. The fetus was returned to the uterus and the uterine incision closed. The catheters were exteriorized through a small incision in the flank sf the ewe, and the abdominal incision of the ewe was closed. Catheters were stored in clean plastic bags tucked under No. 8 tubular elastic net bandage (Medical Mart, Mississauga, Ont.) wrapped around the ewe's abdomen. Postoperative analgesia was provided using 5 mg Numorphen suppositories (Du Bont Chemicals Inc., Scarborough, Ont.). The ewes were housed in metabolic cages, and catheters were flushed twice daily with sterile heparinized saline (20 U/mL) to maimtain patency. Amniotic pressure was recorded continuously on a Grass model 78 polygraph (Grass Instruments, Quincy , Mass.), by coupling catheters to Statham P23 pressure transducers (Gould Inc., Oxnard, Calif.). Radioactive microsphere determinations of blood flow were performed at 120 or 140 days gestation (see below). Surgery was also performed on ewes at 140- 144 days gestation as described above. A catheter was advanced into the left ventricle of the fetus via the left common carotid artery. Position of the catheter tip was determined by coupling the catheter to a pressure transducer and chart recorder and noting when ventricular wave forms were observed. Another catheter was placed in the abdominal aorta via the femoral artery. Arterial pressure was measured daily as described above. When amniotic pressures indicated the onset of labour, catheters were knotted and cut close to their point of exit from the ewe. The ewes delivered lambs vaginally with staff in attendance. Catheters readily passed through the maternal exit site and were delivered with the lambs. They were recoupled to stopcocks. then packaged in the pockets of nylon jackets worn by the lambs. Newborn lambs were fed for at least 1 day by the ewe, to ensure they received coIostmm. Subsequently, they were bottle fed 3 times per day with a commercial ewes' milk replacement. Microsphere measurements of blood flow were performed 3 days after birth (see below).
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Surgery was also performed on 14-day-old lambs. The lambs were anesthetized with 1 - 1.5 % halothane in 0,that was delivered via a face mask. Under sterile conditions, a catheter was inserted retrogradely into the left carotid artery and advanced into the left ventricle, and a second catheter was advanced into the abdominal aorta via the femoral artery. Microsphere measurements of blood flow were performed at 21 days s f age (see below). For most preparations, arterial blosd Pao,, Paco,, and pH were measured every other day from a 1-mL sample of blood using a Corning model 170 pHIblood gas analyzer (Corning Medical and Scientific, Medfield, Mass.). kfeasurernents of blood flow, using mdiocrctdve microspheres Procedures for microsphere determinations of blood flow were adapted from Heyrnann eb al. (197'7). Different injection and withdrawal sites were chosen for fetuses and lambs because of the unique arrangement of the fetal circulation. In the fetus, spheres injected into the inferior vena cava pass via two routes to the systemic circulation (right heart and ductus, or foramen ovale and left heart) to deliver different concentrations of microspheres to the upper and lower body. For this reason, reference samples were withdrawn simultaneously from the fetal carotid and femoral arteries. Postpartum, microspheres in-jected into the left ventricle are delivered in equal concentrations to all systemic tissues, so a single (femoral) reference sample was employed. Hn fetuses, the femoral and carotid arterial catheters were coupled to a Harvard m d e l 8-945 infusion -withdrawal pump (Harvard Apparatus, South Nantick, Mass.). Withdrawal of blood was inif ated at a rate of 2.5 mL/rnin9 then 0.5 X lo6 microspheres/kg body weight (estimated) were injected immediately via the femoral vein catheter. Microspheres were 15 pm in diameter and were labelled with 4 6 S (3M, ~ New Brighton, Minn.). Blood withdrawal was continued for a total of 2.5 min. Additional samples of 0.20 mL of blood withdrawn from each catheter yielded only background radioactivity, confirming that an adequate withdrawal duration had been employed. Thirty minutes after microsphere injection, the ewe and fetus were euthanized by injecting 20 mL of T-61 (2W mglml N-1-2-rn-methoxyphenyl-2-ethylbutyl-(1)1-2-hydroxybutyramide 50 m g / d 4,4 '-methylene-bis(cyclohexylt~e~y1ammonium iodide), and 5 m g / d tetracaine HCl; Homhst Canada Inc.. MontrCal, Que.) into the jugular vein of the ewe. Abdominal and uterine incisions were opened, the umbilical cord was tied, and the fetus was removed. All cotyledons were carefully excised from the utems; they were cut from the fetal side of the utems to ensure that all fetal membranes were included in the sample. Total radioactivity of cotyledons was used to determine placental blood flow. Hn lambs, procedures were as for fetuses except that microspheres were injected into the left ventricle and a single reference sample was withdrawn from the femoral arterial catheter. Thirty minutes after sphere injection, the lamb was sacrificed by injection of 1 mL of T-61 into the femoral artery.
Blood j b w and cardiac output determinations Tissues were carefully excised, weighed, minced, and dried in an oven at 60°C. The tissues were ground in a blender, then poured into plastic test tubes to be counted using a y-counter (Cornpugamma, Pharmacia LKB, Cambridge, England). Blood flows to tissues were determined using the formula (Heyrnann et ab. 1977)
where Q, is blood flow to the tissue sample. Crrepresents counts per minute recorded from the reference sample, Q, is the withdrawal rate of this reference sample in millilitres per minute, and Cs represents the counts per minute recorded from the tissue sample. Blood flows per 100 g tissue to the right and left kidneys, right and left cerebral hemispheres, and to skin on the right and left side of the head were compared to ensure adequate mixing of the spheres. Adequate mixing was confirmed since variability between left and right side blood flows withln animals was much less than variability among
CAN. J. PHYSHBL. PHARMACOL. VOL. 70, 1992
TABLEI . Perinatal blood gases and CO
Pao, (rnmHg) Paco, (mmHg) PH CVO (mE/(min kg)) C 0 (rnL/(min = kg))
20.8+0.6(3) 44.8f 1.4 (3) 7.42f 0.01 (3) 363 f 46 (5)
14.5+1.7(6) 49.4k0.9 (4) 7.39f 0.82 (6) 335 f37 (5)
69.9+3.1(4) 42.0f 3.5 (4) 7.41 k0.804 (4) 412181 ( 5 ) 206 f57 (5)
87.7k0.7 (2) 34.7k0.7 (2) 7.48f0.00 (2) 414116 (5) 207 1 1 (5)
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NOTE: Values are mean f SEM. Numbers in parentheses are sample sizes. dg, days gestation; do. days postpartum.
TABLE2. Perinatal organ blood flows per 100 g tissue wet weight Organ
120 dg
140 dg
3 do
21 do
Cerebral hemisphere Cerebellum Intestine Kidney Liver (hepatic artery) Adrenal Spleen Diaphragm Perirenal fat Skeletal muscle Skin Bone Subdiaphragmatic carcass NOTE: Values are mean k SEM. rz = 6 for 140 days gestation. n = 5 all other data, except n = 4 for skin and cerebral hemisphere at 3 days. dg, days gestation; do, days postpartum. "Significantly different ( p < 0.05) from value measured at 140 days gestation.
animals for all three tissues. Specifically, we found that the standard error between left and right side samples was much less than the among-animal standard error for either sample. This was true for both skin and kidney blood Wows at all ages. There were no obvious outliers. Total organ blood flow was measured, and blood Wow per 100 g tissue wet weight was calculated for several organs, including the placenta (all cotyledons), right and left cerebral hemispheres, cerebellum, kidneys, adrenals, diaphragm, liver and gallbladder, the subdiaphragmatic carcass, and the portion of the intestine supplied by the superior mesenteric artery (see below). In addition, samples of skin from the chest wall, hind-limb muscle and bone, and perirenal fat were excised and weighed, and blood flows per 100 g tissue wet weight were determined. Subdiaphragmatic carcass flow was calculated by counting the tissues of the subdiaphragmatic amnk and abdominal wall, and doubling right hind limb flow measurements. The liver and gallbladder were excised and counted together for determination of hepatic arterial blood Wow. We assume that the diaphragm is supplied entirely via the celiac artery in fetal and neonatal lambs; thus, blood flow was calculated by comparison with the femoral artery reference sample. This measurement is subject to a minor error in the fetal lambs, since a small proportion s f diaphragmatic flow is supplied by the mammary artery, which branches from the aorta upstream of the ductus arteriosus and carries output from the left ventricle. In newborn lambs, cardiac outputs (CO) were calculated using the formula (Heymann ef al. 1977)
where Q,represents the withdrawal rate of the femoral artery reference sample, Ct is total counts per minute injected, and Cr is counts per minute recorded from the femoral artery reference sample. In fetuses, combined ventricular output (CVO) was estimated using the equation
where Qc represents withdrawal rate of the carotid artery reference sample, Ct is total counts per minute injected, Q,is withdrawal rate of the femoral artery reference sample, Cf is counts per minute in the femoral artery reference sample, and Cc is counts per minute in the carotid artery reference sample. This estimate is derived from equation 1, based on the assumption that lower body blood flow is 3 X upper body flow in fetal sheep (Rudolph 1985). It also assumes that counts per minute in blood delivered to the lungs and the Bower body are equal. This latter assumption introduces a small error as a result of the minor contribution of left ventricular output to lower body blood flows.
Data analysis Sample sizes for b l o d Wow measurements were pm = 5 per group for 120 day gestation fetuses and 3- and 21-day-old lambs, and az = 6 for 140 day gestation fetuses. Counts were not recovered for skin and cerebral hemispheres for one 3-day-old lamb, so n = 4 for these data. Dunnett's tests were used 6 0 determine significant differences in blood flow and pressure at different ages. Our primary goals were to test for significant changes in blood flow and pressure during late gestation and to measure changes associated with birth as well as later during the postnatal period. Consequently, blood flows and pressures at 120 days gestation and at 3 and 21 days postpartum were compared with flows and pressures near full term (140 days gestation).
Results B&oodgases and cardiac output Table 1 displays data on blood gases, CO, and estimated CVO in the perinetal period. fio2, Paco2, and pH (Table 1) were in accord with previous measurements from fetal and neonatal blood samples (Richardson et al. 1989). Estimated CVO did not change significantly during late gestation (120148 days) . CVO was slightly, but not significantly, above fetal levels at 3 days and was unchanged at 21 days. We observed no change in CO (left ventricular output) per kilogram body
BENDECK AND LANSILLE
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(A)
Cerebral Hemispheres
Kidneys
Cerebellum 6-%
t
28
1
FIG. 1. Total tissue blood Wow (rnklmin), measured at 120 and 140 days gestation and 3 and 21 days postpartum in (A) right (a) and left ( 3 )cerebral hemispheres and (B) cerebellum. Values are mean k SEM . n = 4 - 6 per group. "Significantly different from value at 140 days gestation, p < 0.05. Vertical broken rule indicates mean gestational age at parturition.
weight between 3 (206 f 57 mL/(min . kg)) and 21 days (207 f 11 mL/(min * kg)) postpartum. Placental blood flow (not shown) was 657 38 rnL/rnin at 120 days gestation and was not significantly different (557 42 mL/min) at 148 days gestation.
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+
'irissue blood jbws
Total tissue blood flows are shown in Figs. 1-3. As in many tissues, brain blood flows increased in late gestation (Fig. 2). Between 120 and 140 days gestation, total right and left cerebral hemisphere blood flow increased by 158% and total cerebellar flow increased by 204 % . At birth, cerebral and cerebellar blood flows decreased dramatically (by 70% between 140 days gestation and 3 days postpartum). Blood flows to brain tissues then increased, but remained below fetal levels at 21 days postpartum. Cerebral hemisphere flows increased by 122%, from 16.1 &- 2.6 mL/rmin at 3 days post2.3 mL/min at 21 days postpartum, and partum to 35.8 cerebellar flow increased by 67%, from 4.68 f 0.91 to 7.8 1 +_ 0.57 mL/min between 3 and 2 1 days postpartum. The same trends were observed for cerebral and cerebellar blood
+
FIG. 2. Total tissue blood flow (mklmin), measured at 128 and 140 days gestation and 3 and 21 days postpartum in (A) kidneys and (B) adrenals. Values are mean 9 SEM. n = 5 -6 per group. "Significantly different from value at 148 days gestation, g < 0.05. Vertical broken rule indicates mean gestational age at parturition.
flows per 180 g tissue (Table 2), primarily because brain weights did not change postpartum. No differences were seen in perfusion of left and right hemispheres at any time, which probably indicates that collateral supply to the left hemisphere totally compensated for catheterization of the left carotid artery in fetuses. Total renal blood flows nearly doubled in late gestation (Fig. 2A), from 27.2 3.1 mL/min at 128 days gestation to 5 1.0 3.6 mL/min at 140 days gestation, largely as a result of increased perhsion per 180 g weight (Table 2). No change in renal perhsion accompanied parturition, but total blood flows again rose dramatically, by 3-fold, by 21 days postpartum. However, half of this increased total blood flow was due to increased renal tissue weight and half was a result of increased relative tissue perfusion, since there was a 3-fold increase in total blood flow but only a 1.5-fold increase in blood flow per 180 g tissue weight (Table 2). A 5-fold increase in adrenal blood flow, from 0.690 f 8.080 mL/min at 120 days gestation to 3.49 f 0.25 mL/min at 148 days gestation, was again due to approximately equal increases in tissue weight and relative tissue perhsion. There was no further significant change in total blood flow at the
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CAN. J . PHYSIOL. PHARMACOL. VOL. 70, 1992
(A)
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intestine
Diaphragm
Subdiaphragmatic Carcass
Hepatic Artery T
FIG.3. Total tissue blood flow (mE/min), measured at 120 and 140 days gestation and 3 and 21 days postpartum in (A) intestines and (B) hepatic arterial circulation. Values are mean -t SEM. n = 5 or 6 per group. "Significantly different fron-n value at 240 days gestation, p < 0.05. Vertical broken rule indicates mean gestational age at parturition.
postpartum times we assessed. although considerable variability between animals was observed (Fig. 2B). Substantial increases in blood flows to the gastrointestinal tract were concentrated in the prenatal period. Intestinal blood flows increased by 4-fold in late gestation (Fig. 3A). Again, tissue growth and increased relative tissue perfusion contributed equally. No changes occurred in the immediate perinatal period, and after birth, a doubling of mean intestinal blood Wows was not statistically significant, as a result of high variability between animals. Hepatic arterial blood Rows to liver and gall bladder also increased 4-fold in late gestation, from 4.10 1.26 rnl/rnin at 120 days gestation to 17.1 f 4.8 rnl/rnin at 140 days gestation (Fig. 3B). but in this case, the increase was due entirely to a 4-fold increase in perfusion per 1100 g tissue (Table 2). No significant increases were recorded at times after 140 days. Splenic blosd Rows showed no significant changes in the perinatal period (Table 2). Diaphragmatic blood flows were surprisingly consistent between fetal preparations, given that no attempt was made to define whether flow measurements coincided with episodes of
+
FIG.4. Total tissue blood flow (mL/min), measured at 120 and 140 days gestation and 3 and 21 days postpartum in (A) diaphragm and (B) subdiaphragmatic carcass. Values are mean f SEM. n = 5 or 6 per group. "Signiiicantly different from value at 140 days gestation, g < 0.05. Vertical broken rule indicates mean gestational age at parturition.
fetal breathing movements. Total diaphragmatic blood Wows doubled in %ategestation, rising from 1.33 k 0.37 rnl/rnin at 728 days to 2.69 f 0.30 mL/min at 140 days, and doubled again between 3 and 2 1 days postpartum (from 3.75 f 0.63 to 8.44 a 1.73 rnL/min) (Fig. 4A), These changes were due to increases in tissue mass, since there were no changes in perfusion per 100 g tissue. Strikingly, no changes in perfusion of diaphragmatic tissues were seen between 148 days gestation and 3 days postpartum, despite the initiation of lung gas exchange (Fig. 4A). Total blood flow to subdiaphragmatic carcass tissues showed relatively modest changes in the perinatal period (Fig. 4B) and only those changes occurring between 120 and 140 days gestation proved significant, although high data variability may have masked increases after birth. Mean flows changed by nearly 2-fold over this time. The only significant change in carcass blood flows per 180 g tissue was a 42 % fall between 140 days gestation and 3 days postpartum (Table 2). Blood flow to bone per 180 g was halved in late gestation (Table 2). These flows showed another dramatic decrease, by 84% after birth, before recovering slightly between 3 and 21
BEWDECK AND EANGIEEE
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days postpartum. Relative perhsion of skin did not change in late gestation, but fell by 88% postparturn before rising to about half of fetal levels by 29 days postpartum. Although mean blood flow per 188 g to perinatal fat doubled between 120 and 140 days gestation, then decreased dramatically by 21 days postpartum, these changes were not significant as a result s f variability between animals in the immediate perinatal period.
Discussion There are dramatic changes in cardiovascular function during the perinatal period. Changes directly associated with birth include closure of fetal vascular shunts (ductus arteriosus, foramen ovale, ductus venosus), increase in systemic and decrease in pulmonary arterial pressure, greatly increased pulmonary blood flow, loss of placental perfbsion, and increased left ventricular output (Heymann st a / . 1981). Our measurements of CVO in fetal sheep and CO in 2 1-day-old lambs were in accord with those of others (Lister et ak. 1999; Klopfenstein and Rudolph 1978; Rosenberg et al. 1984). However, some previous studies have reported large, transient increases in left ventricular output in the lamb from 150 before birth to -480 ml/(min kg) by 0-3 days after birth (Lister et ak. 1979; Klopfenstein and Rudolph 1978). Our measurements of left ventricular output after birth are not consistent with these data. We measured outputs of 280 ml/(min . kg) at 3 days postpartum. These outflows are undsubtedly above fetal levels, since the left ventricle delivers less than half of the 358 ml/(min - kg) of CVO in utero (Rudolph 1985); nonetheBess, they are well below the values reported in the two previous studies. The reason for this discrepancy is unclear. Recent studies with human subjects indicate that left ventricular output is twice fetal levels at 1 h after birth, but output declines to 44% above fetal values by 24 h and then doesn't change over the subsequent 3 days (Agata et a!. 1991). These data are consistent with our finding of moderately elevated left ventricular output at 3 days. In addition to these changes in central circulatory function, there were large changes in peripheral tissue blood flows throughout the perinatal period. However, one of our most striking findings was that no tissues exhibited increased perfusisn per unit tissue mass between late gestation (140 days) and early postpartum life ((3 or 21 days). Previous investigators have reported increases in brain, hepatic arterial, intestinal, renal, splenic, and diaphragmatic blood flows per 100 g tissue when measurements made at 3 - 10 days postpartum were compared with a single prepartum value (Rosenberg ef a / . 1984); however, our data indicate that many of these increases occur during late gestation. This observation has important implications in that it suggests that late gestational growth and maturation of tissues are major determinants of changing tissue perfusion. Late gestational increases in flow to the diaphragm, skin, and muscle largely paralleled tissue growth, and perfusion per 100 g did not change significantly; however, increases in adrenal, intestinal, brain, and renal blood flows exceeded those attributable to growth, and flows per 108 g approximately doubled. Intestinal blood flows may increase in late gestation as a result of the metabolic demands imposed by maximal proliferation of lymphoid cells in Peyer9spatches at this time (Reynolds and Morris 1983), whereas increased brain blood flow probably reflects high levels of cerebral angiogenesis despite slowed brain growth in the perinatal
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period (Bar 1983). Bone blood flows were halved between 128 and 140 days gestation, but the reason for this decrease is unclear. These results underscore the importance of both growth and altered tissue metabolism in influencing late gestational blood flow changes. Our failure to detect increased perfusion at birth to any of the tissues that were examined reflects, in part, increased blood oxygen content after parturition. Thus, Pao2 increased from about 20 to almost 90 mmHg (I mmHg = 133.3 Pa) over the ages we examined, a finding consistent with previous reports (Richardson et a/. 1989; Rosenberg et al. 1984). This increased Pao2 reduces perfbsion demands, at least in terms s f O2 requirements, and this undoubtedly accounts for the postpartum fall in blood flow to skeletal muscle, brain, and adrenal glands. This interpretation is consistent with the data of Iwamoto et al. (1989), who measured decreases in brain, adrenal, carcass, and heart blsod flows per tissue weight after ventilating the lungs of fetuses in utero. Also, Peeters et a&. (1979) increased fetal blood oxygen content by administering hyperbaric oxygen to pregnant ewes and measured decreased brain, heart, and adrenal flows per tissue weight. For some tissues whose blood supply is not determined primarily by gasexchange requirements, like the kidneys, no change in perfbsion occurred at parturition. Other studies have reported no change in renal or gastrointestinal blood flow per weight between birth and 3 days postpartum (Richardson et al. 1989; Aperia et al. 1977; Nakamura er al. 1989). In contrast, a marked fall in skin blood flow at parturition was probably due to a combination of increased O2 levels and a thermoregulatory response to heat loss in the extrauterine environment. Changes in blood flow distribution immediately surrounding birth are also undoubtedly influenced by dramatic changes in plasma concentrations of catecholamines, angiotensin 11, arginine vasopressin, and prostaglandins E and F (Heymann et al. 1981; Mott 1975; Eliot et a / . 1981; Alexander et al. 1972). Thus, for example, Alexander st al. (1972) have demonstrated the capacity of catecholamines to markedly redistribute blsod flows in newborn lambs. Blood flows to many tissues increased between 3 and 21 days postpartum, although no flows attained values that exceeded perfbsion at 148 days gestation. In summary, we have measured marked increases in total blood flow and perfusion per unit weight in many tissue beds during late gestation. These findings underscore the important influence of late gestational growth and maturation of tissues on blood flow distribution. Large decreases in cerebral, cerebellar, adrenal, skin, bone, and carcass perhsion occurred between 140 days gestation and 3 days postpartum, but there were no significant changes in flow to other tissues at birth. Furthermore, there were no increases in blood flow per weight in any tissue between late gestation and 3 weeks of age. Previous inferences that increases in gut and kidney blood flows between 130 days gestation and 3- 10 days postpartum are attributable to the acquisition of postnatal function in these organs (Rosenberg et dl. 1984) are not supported by our data, which indicate that increased perfusion of these organs occurs mainly during late gestation and is not directly associated with birth.
Acknowledgements The authors acknowledge the technical assistance of Doreen LeBlanc. This research was supported by the Medical Research Council of Canada (MRC). M . P. Bendeck is an awardee of an MRC Studentship. B. L. Langille is a Career Investigator of the Heart and Stroke Foundation of Ontario.
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