JOURNALOF APPLIED Vol. 38, No. 6, June

PHYSIOLOGY 1975. Printed

Control

in U.S.A.

of vascular

sheep umbilical

volume

in

circulation

JOHN M. BISSONNETTE Department ofv Obstetrics and Gynecology, University of Oregon Medical

BISSONNETTE, JOHN MI. Control of vascular volume in sheep umbilical circulation. J. Appl. Physiol. 38(6) : 1057-1061. 1975.-Vascular and extravascular volumes were measured using a single-injection, double-indicator technique, in a perfused umbilical circulation in which umbilical artery (Pfa) and umbilical vein (Pfv) pressures could be independently varied. At constant Pfv, when Pfa was raised from 25 to 35 mmHg, vascular volume increased 16.1 rfi 6 ml (mean + SD) from a control value of 113 A= 38.3 ml. Extravascular volume increased 25.2 + 7.7 ml from 128 =I= 53.3 ml. During further increases in Pfa to 65 mmHg, vascular volume increased approximately 10 ml for each 10 mmHg increment, but no change in extravascular volume occurred. At a constant Pfa when Pfv was raised to 15, 20, and 25 mmHg, vascular volume increased 12.2 =t 3.6, 22.2 =t 5.4, and 28 =t 12.5 ml from the measurements at low Pfv. There was no increase in extravascular volume during elevation of Pfv. At the lower values of Pfa (25-35 mmHg) recruitment of previously unperfused channels and distension are seen. Over the higher ranges of Pfa change and during Pfv elevation there is distension of the vascular bed.

School, Portland,

Oregon 97201

opened, then the space measured by an indicator that traverses the capillary wall minus the vascular marker space, that is, the extravascular volume, will increase. During

distension

of the

capillary

bed,

or any

portion

of the

vascular bed, the spaces measured by both the diffusible and intravascular markers would increase. However, during distension the difference between the two markers would not change, that is, the extravascular space would remain constant. Previous experiments (2) using multiple indicators have enabled us to identify a group of small, lipid-insoluble solutes that freely traverse the capillary wall in the umbilical circulation but during a single transit are not transferred to the maternal circulation across the cytotrophoblast. In the present report we have used one of these (glycerol) to measure the ext,ravascular plus vascular volume. METHODS

umbilical artery ment; distension;

THE

VOLUME

and vein multiple

perfused placenta; dilution curves

within the umbilical circulation in the factors governing exchange fetus. We have previously attempted of vascular pressures on this volume

OF BLOOD

important variable tween mother and exarnine the effects

pressures; indicator

recruit-

is an beto (1).

This earlier study suffered from two defects. When outflow pressure was raised, flow was not decreased so that volume was measured with both inflow and outflow pressure elevated, and recruitment could not be distinguished from vascular distension. In addition, we did not take into account the model proposed by Fung and Sobin (6), which would allow for capillary distension as opposed to recruitment of previously unperfused channels during increases of inflow pressure. In the present study we have examined umbilical blood volume under conditions in which inflow (umbilical artery) and outflow (umbilical vein) pressures were changed independently. Maseri et al. (12) have discussed the problem of recruitment versus distension of the capillary bed. The results of their experiments did not allow the distinction to be made; however, in the discussion they suggest an experimental method with which to approach the problem. These authors have suggested measuring both the vascular and extravascular ‘spaces at varied’ inflow pressures, while outflow pressure is held constant. If previously unperfused beds are

The studies were performed on 10 near-term pregnant ewes. The anesthesia, surgical preparation, and technique of umbilical circulation perfusion have been previously described ( 1). In the present studies the larnb was sacrificed with 3-5 ml of a solution containing in each ml 194 mg sodium pentobarbitol, 20 % (vol/vol) propylene glycol, 10 7c (vol/vol) isopropyl alcohol, and the balance distilled water (Euthanol, Trico Pharmaceutical Co., San Carlos, Calif.). This was injected into an umbilical vein and the cord clamped within 5 s so that the placental bed was not exposed to it. After an umbilical artery and its corresponding vein were cannulated, a third catheter was left in the amniotic space and the uterine cavity closed in layers. Dextran (5 % of mol wt 60,000) in Ringer solution was used for the perfusion fluid and was not recirculated. Pressures were measured in the umbilical artery . (Pfa), umbilical vein (Pfv), and amniotic fluid (model P23 pressure transducer, Statham Laboratories, Hato Key, Puerto Rico) and recorded (model M5P polvgraph, Gilson Medical Electronics, Middleton, Wise.). Pressures were balanced to atmosphere at the level of the top of the uterus. Umbilical artery pressure was controlled by the rate of the perfusion pump and umbilical vein pressure by the height of the outflow reservoir and collection rack. When either Pfa or Pfv was changed, the perfusion was maintained at the new level for at least 5 min before volumes were measured. Volumes were measured using a single-injection doubleindicator technique (3). The injectate consisted of 0.45-

1057

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1058

J. 40

a a

a a ? 9 f

‘O

$

5-

a

_ 0 0

a 0

2 s 0

.z z

Fetal

ooooo O0 OO a 00 0 0 a O Ooo, 000 0 a Ooo a Oooo a a

5,746

a

3 -

Wt,

l

.s

0

2,272

a

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BISSONNETTE

TABLE 1. Pressure-volume results listed in the order in which each determination was made

T-1824 l Glycerol 14c O

@a

l

M.

I 50

I

3,125

60

Seconds

1. Single-injection double-indicator dilution curve. Ordinate: concentration expressed as fractional recovery of injected mass per ml. Abscissa: time (s) at which one-half of sample was collected. Pfa = 51 mgHg, Pfv = 5.5 mmHg, umbilical blood flow = 175 ml. min-l. FIG.

4,688

0.65 mg T-1824 and either 5 ,uCi [ 14C]glycerol (sp act 18.5 mCi/mmol, ICN, Irvine, Calif.) or 2.5 ,&i [14C]glycerol (sp act 57 mCi/mmol, New England Nuclear, Boston, Mass.) in 1.0 ml of maternal plasma. A 40-tube movable collection rack (E. A. Iller, Lachine, Quebec, Canada) was started one-half way through the injection period, and the entire outflow was collected at rates that allowed each tube to fill for 0.8-4.0 s. The volume of the outflow catheter was 3.5 ml. One milliliter of the injectate was added to 99 ml of perfusion fluid to serve as a standard. Five to eight volume determinations were made in each experiment. Volumes were measured at Pfa levels of 25, 35,45, 55, and 65 mmHg, while Pfv was 6.0 mmHg or less. Pfa was held constant at 45, 50, or 55 when Pfv was raised to 15, 20, or 25 mmHg. The outflow samples and standards were analyzed as follows : T-l 824 concentrations were measured in a spectrophotometer (model B, Beckman Instruments, Inc., Fullerton, Calif.) at 620 nm in 0.5-ml cuvettes. For 14C measurements 0.5 ml of sample was added to 0.5 ml 30 % trichloroacetic acid and centrifuged at 3,000 rpm for 10 min; 0.2 ml of the supernatant was added to 10 ml of a commercial liquid scintillation fluid (Aquasol, New England Nuclear) and counted for 10 min (Model 3224 Tri-Carb, Packard Instruments Co., La Grange, Ill.). The concentration for each outflow sample (mg/ml for T-l 824 and cpm/ml for [ 14C]glycerol) was divided by the injected mass to obtain a scaled recovery ratio. The recovery ratios were plotted on semilogarithm paper against the time at which one-half of the sample was collected (Fig. 1). The indicator curves were extrapolated and umbilical blood flow and mean circulation times for each indicator calculated, according to the method of R. M. Effros (personal communication). Vascular volume was then obtained from the product of flow and T-1824 mean circulation time, while extravascular volume was measured as the product of flow and the difference between [ 14C]glycerol and T-l 824 mean circulation times.

2,784

2,955

5,966

5,938

5,966

6,080

g

Pfa, mmHg

45 25 55 44 26 35 65 34 44 45 45 46 45 45 55 50 50 51 51 51 51 51 51 25 ?5 44 54 64 56 56 55 55 56 45 36 64 54 24 46 35 24 45 36 26 45 46 65 55 46 35 56 65 36 46 66 55 45 45 46 46 36 26 46 45 35 55 17 46 26

Pfv,

mmHg

Umbilical Blood Flow, ml min-l

Vascular Volume , ml

Extravascular Volume, ml

340 130 420 345 149 260 485 105 145 140 100 150 77 113 182 153 157 175 110 96 170 130 105 85 153 197 229 275 154 122 157 100 148 175 145 265 210 75 185 142 102 320 265 133 151 345 470 400 281 190 420 488 205 300 496 398 320 192 290 300 254 147 307 314 264 412 74 360 212

115 97 119 112 98 107 126 63 75 79 90 74 93 86 87 59 73 58 79 85 52 75 83 137 160 172 179 193 71 95 76 120 79 62 51 85 74 31 64 56 133 157 148 129 166 152 174 165 167 152 178 193 149 170 198 181 159 175 165 152 141 126 155 169 163 186 135 176 154

132 112 137 130 115 135 139 75 92 89 94 88 89 96 88 73 76 70 74 77 71 72 70 120 144 140 146 139 92 95 89 93 96 75 79 80 76 52 81 77 190 249 231 205 232 232 234 220 219 224 211 209 215 213 220 216 173 175 182 178 184 155 186 183 73 77 60 82 72

5 5 5 4 5 5 6 5 7.5 12.5 18.5 3.5 22 17 1 8.5 14 5.5 20 23 2 19 21 5 5 4 5 6 5.5 17.5 5.5 23.5 5 5 5 6 3 5 6 2 5 6 5 5 17 4 6 5 0 5 3 3 3 r ;; 1 6.5 25 17 1 0 0 0 16 4 6 0 5 0

-

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CONTROL

OF

VASCULAR

VOLUME

IN

UMBILICAL

TABLE 2. Summary of increase in vascular and extravascular volumes with Pfa* Pfa,

Increase in vascular volume, t ml Increase in extravascular volume,t ml No. of comparisons * Pfv constant

at 6 mmHg

mmHg

25-35

35-45

45-55

55-65

16.1 zt6 25.2 rt7.7 9

11.0 zt4.3 2.4 rt8.6 14

10.0 zk2.9 0.2 zt5.9 11

12.2 zt3.8 2.5 zt7.0 6

or less.

iValues

are means

1059

CIRCULATION

zfr SD.

RESULTS

Figure 1 shows the indicator dilution curves obtained in these experiments. Appea,rance times of both indicators are similar. [ 14C]Glycerol has a curve in which the peak is less than that of T-1824. The peak time and mean circulation time of [ 14C]glycerol are delayed relative to T-1824. The ratio of the area under the entire [ 14C]glycerol curve to that of T-1824 was 0.93 rt 0.08 (mean =t SD) in 69 determinations. In a single transit very little [14C]glycerol is lost to the maternal circulation. All pressures reported are referred +o amniotic fluid pressure, which ranged from 0 to 3.5 mmHg in these studies. In an individual preparation the flow obtained at a given pressure setting remained consistent, which indicates the stability of the perfusion. The umbilical artery and umbilical vein pressures, umbilical blood flow, and vascular and extravascular volumes are given in Table 1. The values in Table 1 represent changes in the vascular bed of that portion of the placenta served by one umbilical artery and its corresponding vein. The results are listed in the order they were measured in each experiment. Volumes measured while going from a low to a high inflow pressure were the same as returning from a high to a low pressure; that is, no hysteresis effect was seen. This was true for increases in outflow pressure as well. In Table 2 we have grouped the volume changes over four Pfa increases, while Pfv was 6.0 mmHg or less. Umbilical artery pressure referred to amniotic fluid pressure in the awake, unstressed lamb has been reported to be 44 mmHg (3) ; we have used 45 mmHg as our midpoint in studying changes 20 mmHg above and below this value. The mean increases in either vascular or extravascular volume in Table 2 were obtained from the increase observed in each preparation. The vascular volumes show a wide range when one animal is compared to another (Table 1). However, within each individual animal response to an increase in either inflow or outflow pressure is quite consistent. It can be seen that when Pfa is raised from 25 to 35 mmHg, the vascular volume increase of 16.1 r+: 6.0 is somewhat greater than the increase seen over the range between 35 and 65 mmHg. In addition, in this lower range of Pfa change, an increase in extravascular volume is also observed. However, where inflow pressure is raised from 35 to 65 mmHg, no further increase in extravascular volume occurs. The mean and standard deviations of the vascular volume changes within individual preparations when Pfv was raised to 15, 20, and 25 mmHg are shown in Fig. 2. The Pfa level in these measurements was kept constant by lowering umbilical blood flow at the elevated levels of Pfv. Vascular

volume increases at Pfv of 15 mmHg and again at 20 mmHg. No additional increase occurs when Pfv was further elevated to 25 mmHg. There was no consistent change in extravascular volume when Pfv was elevated at constant Pfa (Table 1). DISCUSSION

The vascular volume on the fetal side of the sheep placenta shows a wide variation from animal to animal. Using radioactive iodinated serum albumin Creasy et al. (4) report a value of 48.1 & 35.1 (mean r+ SD) ml/kg of fetal weight in 17 animals. If the values at Pfa 45-50 mmHg and Pfv < 6.0 mmHg in the present studies are doubled and divided by fetal weight, the result is 50.4 =t 11.2 ml/kg (18 measurements in 9 animals). Because of this variation from one preparation to another in this study, the mean volume at each pressure is not reported. Rather the change in volume during pressure changes in each animal is examined. Injections in polyethylene tubing show no separation of the vascular and diffusible indicators and accurately measure the volume of the tubing (J. M. Bissonnette, B. Burns, and G. H. Gurtner, in preparation). In previous studies (1) using a similar preparation, we have reported that there was a significant increase in umbilical vascular volume when umbilical vein pressure was raised above the surrounding pressure. As mentioned, in these studies flow was constant, so that umbilical artery pressure also rose, and therefore recruitment could not be separated from distension of the vascular bed. In two preparations we also measured vascular volume at increasing inflow pressure, while outflow pressure was held constant and found no consistent change in volume. In the earlier studies the fetus had been removed from the amniotic cavity, which is a possible explanation for the discrepancy with the present report. The increase in both vascular and extravascular volume when inflow pressure is raised from 25 to 35 mmHg is consistent with the perfusion of channels that are closed at a lower inflow pressure (12). It is unlikely that the increase in extravascular volume represents pericapillary edema since this effect would be seen especially at the higher inflow pressures. However, in raising Pfa from 25 to 35 mmHg a

-

I

IO

I

I

I

I

15

20

25

30

PFV

m m Hg

FIG. 2. Change in vascular volume at increased Pfa constant. Numbers in brackets refer to number

levels of Pfv wi th of comparisons.

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1060

J. M.

TABLE 3. Cafiillary in which [Vjglycerol Fetal

Wt, g

5,746 2,272 4,688 2,748

5,966

Pfa, mm&

44 26 34 44 54 64 56 56 55 55 56 36 26

volume changes in determinations recovery equals T-1824 recouery Pfv, mmHg

4 5 7.5 5 6 5.5 17.5 5.5 23.5 5 5 5

[14C] Glycerol 1824

0.99 0.99 1.00 1 .Ol 1.00 0.99 .99 1.01 1 .Ol 1 .oo 1 .oo 1 .Ol 1 .Ol

Capillary Volume, ml

Vascular Volume, ml

45.4 32.9 22.8 42.3 3’3.6 44.9 26.2 49.1 24.3 45.1 24.7 79.5 62.4

112 98 63 75 179 193 71 95 76 120 79 148 129

portion of the increase in vascular volume may be caused by distension as well as recruitment. The mean values for vascular volume and extravascular volume at Pfa 25 are 113.1 ml and 127.6 ml (8 measurements in 6 animals). If one-third of the vascular volume is capillary volume (see Table 3 and below), then the ratio of capillary volume to extravascular volume would be 37.7 : 127.6 or approximately 1: 3.4. If all of the vascular volume increase (16.1 ml) seen when Pfa is raised from 25 to 35 mmHg were due to recruitment, then extravascular volume would increase by 3.4 times this value, assuming that the extravascular space surrounding recruited capillaries is similar to that surrounding the capillaries open at Pfa 25 mmHg. However, the observed increase in extravascular volume (25.2 ml) is only 1.5 times the increase in vascular volume. Thus over the range of Pfa 25-35 mmHg both recruitment and distension appear to be taking place. Power and Longo (13) h ave shown that the umbilical circulation follows the principles of a Starling resistor. We have previouslv , demonstrated (1) that when neither amniotic fluid pressure nor maternal vascular pressure in the placenta is elevated, the surrounding pressures exist in extracotyledonary fetal vessels, that is, vessels that are not in intimate contact with maternal vessels and are not subject to changes in maternal vascular pressure. The present studies are consistent with the view that these fetal vessels have a spectrum of critical opening pressures. The increase in vascular volume, but stable extravascular volume, either with independent increase of inflow pressure from 35 to 65 mmHg or with an increase in outflow pressure at constant inflow pressure, suggests vascular distension. However, it would not seem to distinguish distension in large vessels (arteries and veins) from a distension of the capillary bed. The model of Fung and Sobin (6) predicts that the capillary sheet will distend at increased inflow pressures but is unresponsive to changes in outflow pressure. This model is based on sheet flow in the pulmonary circulation and may not apply to the cotyledonary placenta. Goresky (7) has suggested a method of determining capillary blood volumes, using the double-indicator technique. This is based on the assumption that delay in mean transit of the diffusible indicator relative to the vascular

BISSONNETTE

indicator is proportional to the volumes that each reaches. During flow through nonexchange vessels there is no separation of transit times. Thus the ratio, mean transit time for the diffusible indicator minus the transit time through nonexchange vessels to mean transit time for the vascular indicator minus the transit ’ time through nonexchange vessels, is proportional to the ratio of the volume of distribution of both indicators. A value for this later ratio can be obtained from the peak recoveries of both indicators. Since the transit times of both indicators are known, a value for transit through nonexchange vessels is obtained. From this value and the vascular indicator mean transit time, capillary transit time is calculated. Capillary volume is obtained from flow and capillary transit time. The method depends upon complete recovery of the diffusible indicator, which was not the case in all of our studies. We have calculated capillary volumes in those experiments where the recoverv of [ 14C]glycerol was essentially that of T-l 824, and these are pre. sented in Table 3. These calculations suggest that elevation of either inflow or outflow pressure causes distension of the capillary bed in the umbilical circulation. In a subsequent report Goresky and Silverman (8) have shown that the effect of the outflow catheter causes an overestimation of capillary volume. This error is greatest at high flow rates, so that the increase in capillary volume observed at elevated inflow pressure may be in part due to this effect. However, at elevated outflow pressure flow is decreased, and we still observed an increase in capillary volume. The volume of our outflow catheter was small (3.5 ml) and our flow rates well below the 800 ml min-l, at which Goresky and Silverman noted capillary volume was overestimated by 15 %. a value of 10 ml for Longo et al. (11) h ave obtained capillary blood volume in the sheep umbilical circulation. They measured placental carbon monoxide diffusion at various levels of oxygen partial pressure. Thus this smaller value is a measurement of that capillary volume that exchanges with the maternal circulation. The rneasurements we have made using the double-indicator dilution technique are a,n estimate of those vessels in the umbilical circulation in which there is exchange into an extravascular space. In addition, the method used by Longo and co-authors is based on the assumption that placental diffusing capacity for carbon monoxide remains constant at elevated oxygen tensions. Recent studies (9, 10) have suggested that placental oxygen transfer involves facilitated transport. If carbon monoxide and oxygen compete for the same placental carrier, then the carbon monoxide diffusing capacities at high oxygen tension would be decreased giving a low value for capillary volume. While there is a wide variation (15-50 %), the capillary volumes in Table 3 represent approximately 30 % of the placental vascular volume. As mentioned above the double indicator tends to give an erroneously high value for capillary volume. However, 10 ml for an entire fetal placental capillary volume would represent well under 10 % of the blood volume on the fetal side of the placenta. Both inflow and outflow pressures in the umbilical circulation will have an effect on the exchange area of the fetal side of the placenta. This study indicates the magnitude of changes within the vascular and capillary bed, but the auantitative changes in fetal maternal exchange would have

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CONTROL

OF

VASCULAR

IN

VOLUME

UMBILICAL

to be established by studying the placental pacity at varied inflow and outflow pressures. The animal

author appreciates preparations.

the

assistance

of J. Eugene

diffusing Welch

1061

CIRCULATION

ca-

with

This ciation.

study was supported Dextran was made

by a grant from the available by Pharmacia,

Oregon Heart AssoUpsala, Sweden.

the Received

for

publication

12 July

1974.

REFERENCES BISSONNETTE, J. M., AND R. C. FARRELL. Pressure-flow and pressure-volume relationships in the fetal placental circulation. .J. AppZ. Physiol. 35 : 355-360, 1973. BISSONNETTE J. M., M. HAAS, AND R. C. FARRELL. Permeability of water and non-electrolytes in the sheep placenta. Physiologist 16: 266, 1973. CHINARD, F. P., AND L. B. FLEXNER. Capillary permeability. BUZZ. Johns Hopkins ffos$~. 88 : 489-492, 195 1. CREASY, R. K., M. DROST, M. V. GREEN, AND J. A. MORRIS. Determination of fetal, placental and neonatal blood volumes in the sheep. FABER,

Circulation Res. 27 : 487-494, 1970. J. J. Regulation of fetal placental

Gas Exchange and H. Bartels. and Welfare, FUNG, culation

and Blood Flow in the Placenta, Bethesda, Md. : Department 1972, p. 157-173.

Y. C., AND S. S. SOBIN. Pulmonary Res. 30 : 470-490, 1972.

blood

flow.

In : Respiratory

edited by L. D. Longo of Health, Education, alveolar

blood

flow.

Cir-

C. A. A linear method for determining liver sinusoidal 7. GORESKY, and extravascular volumes. Am. J. Physiol. 204: 626-640, 1963. 8. GORESKY, C. A., AND M. SILVERMAN. Effect of correction of catheter distortion on calculated liver sinusoidal volumes. Am. J. Physiol. 207 : 883-892, 1964. 9. GURTNER, G. H., AND B. BURNS. Possible facilitated transport of oxygen across the placenta. Nature 240 : 473-475, 1972. 10. GURTNER, G., AND B. BURNS. Further evidence for a specific 02 carrier in the placenta. Physiologist 16: 331, 1973. L. D., G. G. POWER, AND R. E. FORSTER II. Placental 1 l. LONGO, diffusion capacity for carbon monoxide at varying partial pressure of oxygen. J. A#Z. Physiol. 26 : 360-370, 1969. 12 MASERI, A., P. CALDINI, P. HARWARD, R. C. JOSHI, S. PERMUTT, ’ AND K. L. ZIERLER. Determinants of pulmonary vascular volume. Circulation Res. 3 1 : 2 18-228, 1972. 13. POWER, G. G., AND L. D. LONGO. Sluice flow in placenta: maternal vascular pressure effects on fetal circulation. Am. J. Physiol. 225: 1490-1496, 1973.

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