Cerebral blood flow, oxygen consumption, and glucose utilization of fetal lambs in utero

Cerebral blood flow, oxygen consumption, glucose utilization

of fetal lambs in utero

EDGAR

M.D.

JACK

L. M.

SCHNEIDER,

NICHOLAS JAMES

MAKOWSKI,

G. R.

Denver,

TSOULOS,

C.

GIACOMO

M.D.

COLWILL,

FREDERICK

and

M.D. M.D.

BATTAGLIA,

MESCHIA,

M.D. M.D.

Colorado

Cerebral blood jlow and the distribution of cephalic fZow were measured by the microsphere technique in 12 fetal lambs in utero one hour postoperatively and in I I fetal lambs in utero 24 to 72 hours postoperatively. The mean fetal cerebral blood flow for the two groups was 111.0 i 7.8 and 120.6 ? 8.7 ml. per 100 Gm. per minute, respectively. The mean distribution of cephalic flow 24 to 72 hours postoperatively was brain 37.6 per cent f. 1.8, eyes 4.0 per cent f 0.3, skin 12.3 per cent 2 0.7, and remainder of tissue 46.1 per cent + 1.5. In 8 fetuses, the mean cerebral oxygen consumption was 4.04 ? 0.17 ml. per 100 Gm. per minute 24 to 72 hours postoperatively. Simultaneous fetal cerebral glucose and oxygen consumptions were estimated in 4 fetuses. The mean cerebral glucose consumption was 5.31 f 0.42 mg. per 100 Gm. per minute, and the mean glucose-oxygen quotient was 1.10 with 95 per cent confidence limits 0.95 and 1.25. The results indicate that these uptakes are comparable to those observed in the brains of adult animals.

cliniT H E R E L A T I 0 N s H I P between tally recognizable fetal distress and later neurologic handicap has been amply documented in the clinical literature. Recently Amiel-Tisonl reported data relating neurologic damage to obstetric or neonatal complications. Neurologic damage from various complications of pregnancy may be avoidable as we gain more understanding of the factors regulating fetal cerebral metabolism.

There have been few investigations on the regulation of fetal cerebral blood flow and metabolism. Previous studies on fetal cerebral blood flow have been performed in a variety of ways: as part of a study on the distribution of fetal cardiac output,” on exteriorized fetuses,3 or finally by attemptin,q to relate flow determined in a.single carotid artery to cerebral flow.‘, 5 There is less information about cerebral metabolism in fetal life. In the present investigation, we have developed an animal preparation for the study of fetal cerebral blood flow and metabolism in utero. A description of this method and the experimental results thus far obtained form the substance of this report,

From the Division of Perinatal Medicine, Departments of Obstetrics and Gynecology, Pediatrics and Physiology, University of Colorado Medical Center. This work was supported in part by Atomic Energy Commission Grant AT (I l-1)-1 762 and United States Public Health Service Grant HD 00781. Presented by invitation fifth Annual Meeting Gynecological Society, Virginia, May 18-20,

Materials

Twenty-three ewes of Dorset The gestational

at the Ninetyof the American Hot Springs, 1972. 292

and

methods

fetal lambs from twenty-two or mixed stock were studied. ages ranged from 127 to 150

Volume Number

114 3

Fetal

!I : -/

Innominate

cerebral

blood

Sampling

flow

and

metabolism

293

Syringes

Artery

Fig. 1. Fetal cerebral blood flow. Experimental days as estimated from the breeding record. Prior to the operation, the ewes were starved for 48 to 72 hours and given water ad libitum. The day of operation, they were sedated with pentobarbital (5 mg. per kilogram intravenously) and given spinal anesthesia (6 mg. of tetracaine hydrochloride in hypertonic glucose). Surgical preparation (Fig. 1). The lower anterior abdominal wall of the maternal ewe was incised, and a fetal hind limb was identified through the uterine wall. The uterus and fetal membranes were incised for a distance of 3 cm. over the hoof of the hind limb, and the leg was delivered to the level of the knee. The pedal vein was identified through a skin incision, and a siliconized polyvinyl catheter (0.58 mm. inside diameter) was inserted for a distance of 8 inches. The fetal skin and uterine incisions were separately closed with a continuous 3-O silk suture. A 5 cm. incision was then made through the uterine wall and fetal membranes over a fetal acromioclavicular joint, and the forelimb and shoulder were delivered. The edges of the uterine incision were cauterized in order to obtain hemostasis. The shoulder was incised for a distance of 2 cm. just medial to the acromion and superior to the clavicle. The transverse scapular artery was identified, and a siliconized polyvinyl catheter (0.58

schema.

mm. inside diameter) was inserted for a distance of approximately 4.0 cm. This placed the tip of the catheter just inside the innominate artery without obstructing the blood flow through the carotid artery. The fetal skin incision was closed with skin clips, and the forelimb was replaced. The opposite transverse scapular artery was catheterized in a similar fashion. Facial vein catheterization. After the forelimbs were replaced, the fetal head was delivered in 12 animals and placed in a salinefilled rubber glove. A 2 cm. transverse incision was made about 2 cm. medial to the anterior angle of the eye, and the deep facial vein which communicates via the ophthalmic vein with the cavernous sinus6 was identified. A siliconized polyvinyl catheter (0.38 mm. inside diameter) was inserted into the facial vein for a distance of 2.5 cm., and the skin margins were approximated with tissue adhesive. The fetal head was replaced, and the rubber glove was removed. The edges of the fetal membranes were pulled together and tied around the catheters. The uterine waII incision was closed with a continuous 3-O silk suture, and these animals were studied one hour after the abdominal incision was closed with the animal still on the operating table. Sagittal sinus catheterization. The acute experiments made it apparent that a more

294

Makowski

et al.

representative site than the facial vein was required for sampling the cerebral venous drainage because of the arteriovenous shunting. Hence, in subsequent experiments a sagittal sinus catheter was substituted for the deep facial vein catheter in 11 fetal lambs. To catheterize the sagittal sinus, the fetal scalp was brought to the uterine incision without delivering the nose or mouth, and the skull was exposed in the midline about 1 cm. posterior to the brow through a 1 cm. median scalp incision. A periosteal elevator was used to expose the dura, and a siliconized polyvinyl catheter (0.38 mm. inside diameter) was inserted 2 to 3 cm. through a 22 gauge needle hole. Gentle pressure was applied with a cotton tip applicator to the needle hole of the dura to stop the free flow of blood. The catheter was directed posteriorly so that its tip was at or near the confluens sinus and cemented at its entry through the dura with a drop of tissue adhesive. Bone wax (Ethicon) was used to fill the bony defect and the scalp margins approximated with interrupted 3-O silk sutures. The catheter was secured to the scalp with a 3-O silk suture and a drop of tissue adhesive. This technique was successful in two thirds of the attempts. The failures were due to puncture of either the lateral or posterior wall of the sagittal sinus with the catheter tip. All catheters (pedal vein, both transverse scapular arteries, and sagittal sinus) were exteriorized through a subcutaneous tunnel and placed in a pouch on the ewe’s flank. The animals were standing and eating within 6 hours after operation. Postoperatively, the sheep was kept in a cart containing an adequate supply of hay, grain, and water. These animals were studied 24 to 72 hours postoperatively. Penicillin (600,000 U. of aqueous procaine) and streptomycin (0.5 Gm.) were administered intramuscularly prior to operation and on 3 subsequent days. Microsphere method. The principle and practical application of the microsphere method used in our laboratory for determining organ blood flow have been described previously.7 The distribution of cephalic flow and fetal

Am.

October 1, 1972 J. Obstet. Gynecol.

cerebral flow were measured either one hour following operation or 24 to 72 hours postoperatively. Plastic microspheres labeled with CrS1 (200 mc.) or Cel*l (100 mc.) with a mean diameter of 15 ,LLwere used. All fetuses were given 1,000 units of heparin intravenously prior to the infusion of microspheres. The microspheres were infused into the fetal hind-limb vein over a period of 2 minutes, and the withdrawal time, which started a few seconds before the infusion, for each transverse scapular arterial sample was 4 minutes. The rate of withdrawal by a Harvard pump* was 1.086 ml. per minute. Integrated facial vein or sagittal sinus samples were obtained manually with plastic syringes (Fig. 1) . At the end of the experiment, the mothrr and fetus were killed; the fetus was removed from the uterus and decapitated at the base of the skull. The cranial vault was opened, and the brain was removed and weighed. The skin and eyes were separated from the remainder of the cephalic tissues, and each was separately weighed. The cephalic tissues, including the cranium, were ground twice in a Hobart grinder (Model 4332) and then homogenized in a Waring blendor (Model CB-5) t after the addition of 1,500 ml. of isotonic saline. The fetal brain was homogenized separately in a Waring blendor (Model 1163) after the addition of 100 ml. of isotonic saline. The cephalic tissues were homogenized at 20,000 r.p.m. for 20 minutes, and the brain, at 2,000 r.p.m. for 10 minutes. To obtain representative samples of homogenate: the bottoms of the blenders were equipped with a spout which could be opened while the homogenizer blades were still in motion. Twenty aliquots from the cephalic tissue homogenate and 12 aliquots from the brain homogenate were collected directly into counting vials. The vials were weighed before and after filling. The average weight of an aliquot was 8 Gm. The microspheres of the aliquots and blood samples were collected at the bottom of the vial by centrifu‘Harvard tWaring.

Apparatus Products

Co., Div.,

Millis, New

Massachusetts. Hartford,

Connecticut.

Volume Number

114 3

Fetal cerebral

gation at 2,000 r.p.m. for 10 minutes. The eyes were placed into separate preweighed vials, and the entire cephalic skin was cut into fragments and also placed into previously weighed vials. After the addition of the tissues, the vials were weighed again. The amount of radioactivity in each vial was determined with an automatic gamma scintillation counter with a well-type detector chamber (Nuclear-Chicago, Model 4223”). The total counts per minute for each homogenate were calculated from the mean counts per minute per gram in the aliquots and the total weight of the homogenate. The fraction of cephalic flow to each tissue group (skin, eyes, brain, and remainder of cephalic tissues) was calculated as the ratio of the total radioactivity in one tissue group to the total radioactivity in the sum of the 4 groups. The total radioactivity in the fetal brain was used to calculate total cerebral flow according to the following equation: Blood flow (ml./min.) = total counts per minute in brain x 1.086. total counts per minute in arterial sample (1) Blood measurements on fetuses 24 to 72 hours postoperatively. The fetal arterial and venous blood pressures, arterial microhematocrit, and pH were determined just before and after a cerebral blood flow measurement in all fetuses. The pH was measured with a glass electrode at 39.5’ C. on 0.1 ml. blood samples collected in a capillary tube. In 6 fetuses, the arterial and venous PO, and PcoZ were determined at 39.5’ C. before and after cerebral flow measurement on 1.2 ml. blood samples drawn in 2.5 ml. glass syringes lubricated with silicone oil. The dead space of the syringes was filled with heparin-fluoride solution (1 per cent NaF in heparinized saline). The PO, was measured with a Clark-type electrode, and the Pco~, with a Severinghaustype electrode. An average of 6 sets of arterial and venous o,xygen contents were determined by gas chromatography* in 8 fetuses before, during, and after cerebral flow measurement. Blood samples for oxygen content analysis *Nuclear-Chicago

Corp.,

Des

Plaines,

Illinois.

blood

flow and metabolism

295

were collected anaerobically in heparinized, dry 0.3 ml. capillary tubes. An average of 6 arterial and venous blood glucose samples were analyzed in duplicate by the glucose oxidase method in 4 fetuses. Two 0.1 ml. aliquots of blood were deproteinized immediately with zinc sulfate and barium hydroxide and then centrifuged. The supernatants were analyzed within 6 hours, The mean arteriovenous differences of glucose and oxygen were calculated from the set of arterial and venous blood concentrations expressed as millimoles per liter of blood. The glucose/oxygen quotient8 was calculated as follows: 6 x A glucose

= glucose/oxygen

quotient.

(2)

a oxygen This quotient represents the fraction of the fetal cerebral oxygen consumption required for aerobic metabolism of the cerebral glucose utilized. Fieller’sQ theorem was used to calculate the mean values and 95 per cent confidence limits of this ratio. Results Fetal cerebral blood flow and distribution of cephalic flow: Acute studies. The fetal weight, brain weight, counts per minute in the brain, mean arterial counts per minute, facial vein counts per minute, brain flow, and the distribution of cephalic flows are presented in Table I. The mean brain flow, calculated by assuming no venous loss of microsphere, was 111.0 + 7.8 ml. per 100 Gm. per minute with a range of 58 to 179 ml. per 100 Gm. per minute. The mean per cent distribution of cephalic flow to the brain was 44.0 + 2.9 of the total. In 7 fetuses, cerebral blood flow measurements were repeated one hour after the initial determination. In each instance, the cerebral blood flow increased. The mean per cent increase in cerebral flow was 19.9 t 4.1. With the increase in cerebral flow, there was a concomitant decrease in the relative distribution of cephalic flow to other parts of the head. Studies 24 to 72 hours after operation. The gestational ages, fetal sex, and fetal and brain weights are presented in Table II. The

296

Makowski

et al. Am.

Table I. Acute cerebral Animal No.

flow and distribution Gestational (days)

age

of cephalic

Fetal weight (cm.)

October 1, 1972 J. Obstet. Gynecol.

flow Brain weight (Gm.)

Total

c.p.m. brain

Mean

arterial c.p.m.

42*

135

2,106

49.7

10

135

3,922

55.2

296,232 313,484

5,275 5,068

S-24*

135

2,886

54.7

405,83 1 130,738

9,032 2,673

x-21

137

4,626

56.7

518,880 732,000

454*

137

2,506

49.5

2,643,107

34,517

33

140

3,650

50.5

493,922 584,848

7,010 7,015

43

141

3,768

58.9

778,975 777,228

15,301 12,532

x-2

142

4,242

52.7

210,879

4,213

X-19”

147

4,222

50.9

465,647

10,627

x-3

148

3,650

49.6

205,196 154,346

7,786 4,349

S-25*

150

4,056

59.8

220,419 569,030

3,564 6,857

S-8

150

4,600

63.2

1,488,854

18,339

=

Counts

94,760

10,867 13,906

54.3 t1.3

Mean + S.E.M. c.p.m.

2,509,517

in

per

minute.

*Twins.

total counts per minute in the brain, mean arterial counts per minute, sagittal vein counts per minute, brain flow, and the distribution of cephalic flow are given in Table III. In 13 experiments, integrated samples were obtained simultaneously from both transverse scapular arteries. The mean per cent difference in total radioactivity of the arteries was 8.6 t 2.4. The per cent of radioactivity shunted across the cerebral circulation was only 0.12 + 0.06 for microspheres 15 p in diameter. The mean brain flow was 120.6 + 8.7 ml. per 100 Gm. per minute with a range of 84 to 177 ml. per 100 Gm. per minute. The mean per cent distribution of cephalic flow to the brain was 37.6 f 1.8. In 2 fetuses (W-25 and W-A) another estimate of cerebral blood flow was repeated 24 hours later. There was a 69 per cent increase in cerebral blood flow in Fetus W-25 and essentially no change in Fetus W-A. The mean arterial pressure, hematocrit,

and arterial pH before and after cerebral blood flow measurement are presented in Table IV. Differences were tested for statistical significance by paired analysis. In 6 experiments, the arterial PO, and Pcoz were determined before and after flow measurement (Table IV). The mean arterial PO, before flow was 21.4 rtr 1 .O mm. Hg and after flow, 19.8 k 0.8 mm. Hg. The difference is of borderline significance (p < 0.05). The mean arterial Pcoz before and after cerebral blood flow determination were not significantly different (p > 0.5). The arterial and venous 0, contents and cerebral 0, consumptions measured in 10 experiments are presented in Table V. The mean arterial and venous 0, contents were 3.42 + 0.20 and 1.95 t 0.17 mM per liter, respectively. The mean cerebral 0, consumption was 4.04 + 0.17 ml. per 100 Gm. per minute. The mean arterial and venous glucose con-

Volume Number

Facial

114 3

Fetal

Arteriovenous difference (%)

vein

c.p.m.

-

-

Brain flow (ml./100 Gm./ min.)

cerebral

Distribution Brain

blood

flow

of cephalic

Eyes

and

flow

metabolism

297

(%)

Skin

Remainder

58

62

5

6

27

110 122

35 51

3 4

11 8

51 37

89 97

36 51

4 3

15 9

45 37

45 47

3 3 -

10 9

42 41

303 144

2.8 1.0

91 101

442

1.3

168

89 96

1.3 1.4

15” 179

43 54

4 5

9

3

44 38

377 1,060

2.5 8.5

94 114

50 62

7 5

9 6

34 27

314

7.5

103

33

3

15

49

183

1.7

93

42

4

8

46

84 93

1.1 2.1

58 78

15 23

1 2

24 16

60 59

112 151

39 52

5 5

9 6

47 37

140

52

5

9

34

111.0 27.8

44.0 3.9

4.0 fO.3

10.1 21.1

-

2.8 k0.8

Table

II. Vital

statistics

Sheep No.

Gestational (days)

D-5 D-19A* 19B* D-22? w-22 W-25 W-24 D-28 D-15 D-20 W-A D = Dorset; “Twins.

41.9 +‘7 ‘) --.‘.

age

127 135 135 136 137 137 137 138 139 140 150

Fetal

F M F M F F F M F F F

sex

Fetal weight (Gm.)

2,950 3,254 2,642 2,389 3,252 4,344 4,984 2,630 3,130 3,588 3.766

Brain weight (Gm.1

44.8 47.5 45.4 48.9 50.6 50.9 57.9 47.5 54.5 57.5 49.8

W = Western

tTriplets.

centrations, cerebral glucose consumptions, and cerebral glucose/oxygen quotients in 6 experiments are presented in Table VI. The mean arterial and venous glucose concentrations were 0.923 + 0.100 and 0.691 f 0.109 mM per liter, respectively. The mean cerebral glucose consumption was 5.31 + 0.42 mg. per

100 Cm. per minute, and the mean cerebral glucose/oxygen quotient was 1 .lO with 95 per cent confidence limits 0.95 and 1.25. Comment In contrast to man, the brain of adult sheep is almost entirely supplied by the inter-

298

Makowski

October

et al.

Am. J. Obstet.

nal maxillary arteries which arise from the external carotids. Only the distal portion of the medulla oblongata receives its blood supply from the vertebral arteries. In sheep, these arteries do not anastomose with the circle of Willis, and the internal carotids are

Table III. Cerebral postoperatively

flow and distribution

obliterated.lO However, in fetal lambs, the internal carotids are patent,3 and the fetal brain receives its blood supply from the internal carotids and vertebral arteries. This anatomic arrangement makes the use of electromagnetic flow probes for direct measurement

of cephalic

flow 24 to 72 hours

ArterioveAnimal No. D-5

Total c.$.m. in brain

Mean arterial c.p.m.

171,588

Sagittal vein c.p.m.

3,885

Brain flow (ml./100 Gm./min.)

?lOUS

difference (%I

0

0

1, 1972 Gynecol.

107

Distribution

of cephalic

flow

(To)

Brain

Eyes

Skin

Remainder

36

5

11

48

D-l 9A’

4,688,423

90,840

D-19B

1,905,405

38,956

0

0

117

D-22

2,826,595

64,805

0

0

97

w-22

503,590

10,966

34

0.3

99

34

5

16

45

15,125

-

84

30

5

10

55

87 147

36 36

4 4

13 14

47 46

W-24

676,292

W-25

902,442 700,524

118

22,209 10,200

0.5 0.1

111 10

D-28

7,767,519

173,960

D-15

7,516,024

84,486

0

0

102 177

D-20

3,757,711

66,921

0

0

106

43

3

11

43

W-A

542,684 1,461,592

7,083 19,859

0 60

0 0.3

167 160

41 45

3 3

11 12

45 40

120.6 58.7

37.6 +1.8

4.0 kO.3

12.3 +0.7

46.1 21.5

0.12 to.06

Mean t S.E.M. *Twins.

Table IV. Mean

Animal No. D-5 D-19A D-19B D-22 w-22 W-25 W-25 W-24 D-28 D-15 D-20 ;:; Mean * S.E.M. P

arterial

Postoperative hr. 72 48 48 48 48 48 72 48 48 48 48 24 48

pressure,

hematocrit,

Brain flow (ml./100 Gm./ min.)

and arterial

respiratory

Mean arterial pressure (mm. Hd Preflow ( Postflow

107 118 117 97 99 87 147 84 102 177 106 167 160

50 55 46 54 56 57 50 64 58 54 62 48 46

56 55 50 62 64 58 53 60 54 56 60 48 52

120.6 ? 8.7

53.8 +1.6

56.0 f1.3
gases Hematocsit (%) Preflo w 43 48 44 32 47 45 44 50 1; 44 33 33 42.1 +1.6

Postflow 39 45 43 30 47 44 42 z! 39 44 33 33 40.8 k1.6 <0.005

Volume Number

114 3

Fetal

PH

7.350 +0.004

flow

Arterial PO2

Arterial

7.35 7.33 7.35 7.37 7.35 7.37 7.34 7.35 7.38 7.36 7.33 7.34 7.33

blood

(mm.

POStpOW 7.36 7.33 7.36 7.39 7.31 7.37 7.31 7.34 7.35 7.36 7.34 7.34 7.33 7.345 +0.006 <0.4

PrefEow

22.3 23.6 24.1 -

and

metabolism

299

flows were 71.8 f 2.8 and 15.2 + 0.2 ml. per 100 Gm. per minute, respectively. These flows are significantly lower than the cerebral blood flows measured in the present study (p < 0.001) . It seems likely that at least part of this discrepancy is due to differences in the physiologic states of the two preparations. The lambs used in this study differed from the exteriorized preparation with respect to body temperature (40° C. versus 38’ C.), PcoZ (54 versus 40 mm. Hg) and blood pressure (55 versus 70 mm. Hg) . The mean cerebral blood flow observed by us (121 ml. per 100 Gm. per minute) is much higher than the cerebral blood flow of adult animals. The blood flow to the human brain is 53 ml. per 100 Gm. per minute.*’ Reliable data about the cerebral blood flow of adult sheep are not yet available, but preliminary experiments in our laboratory indicate a magnitude similar to that of the adult human brain. The much higher cerebral flow of fetal life is probably due to the fact that fetal carotid blood is much less oxygenated and has a higher PcoZ than the normal arterial blood of adult animals. The measurements of fetal cerebral oxygen and glucose uptakes presented in this paper represent the first estimates of these variables in nonexteriorized fetuses. The results indicate that these uptakes, expressed as rates

of fetal cerebral blood flow impractical. Carotid blood flows have been measured in fetal and newborn lambs by means of electromagnetic flow probes,4, 5 and inferences have been drawn from these measurements concerning cerebral blood flow. It can be seen from our data that the cerebral blood flow of fetal lambs is approximately 40 per cent of the cephalic flow. This fact and the existence of marked differences in the regulation of flows to different organs and tissues of the body suggest that changes in carotid flow may not be a reliable index of changes in cerebral flow. Direct estimates of cerebral blood flow in fetal lambs have been obtained by Purves and James3 and Rudolph and Heymann. Rudolph and Heymann estimated cerebral blood flow as the percentage of total cardiac output by a microsphere method. Their mean value of cerebral blood flow in term fetuses (132 ml. per 100 Gm. per minute) is quite similar to that obtained in the present study, but the two means are not comparable because of a significant difference in the variance of the two observations (F = 5.3, p < 0.05). Purves and James3 measured arterial blood flow on exteriorized fetuses by the xenon clearance technique. This method estimates separately gray and white matter flows. In their study, the average gray matter and white matter

PWflOW

cerebral

Arterial Pcor Hd

1

(mm.

H.4

Postflow

PrefEow

19.4 19.7 23.0

54.9 51.4 51.7

61.2 51.6 51.9

51.5 52.5 51.6 -

-

20.7 17.3 20.4

19.7 17.3 19.9

55.5 56.5 53.0

21.4 k1.0

19.8 20.8 <0.05

53.8 to.9

)

Postflow

53.4 21.6 >0.05

300

Makowski

Table

October 1, 1972 Am. J. Obstet. Gynecol.

et al.

V. Cerebral

oxygen

consumption 0,

arterial

content

01 venous

(mean)

content

0,

arteriovenous content

(mean)

(mean)

Cerebral 0, consumption (ml./1 00 Gm./min.)

Animal No.

Sampling

D-5

Preflow

7

3.17

0.092

2.07

0.082

1.10

0.078

2.64

D-19B

Preflow Postflow

5 3

4.38 4.63

0.120 0.057

2.67 2.91

0.106 0.052

1.71 1.72

0.094 0.057

4.48 4.51

D-22

Preflow

6

3.33

0.068

1.48

0.066

1.85

0.112

4.02

w-22

PreAow

7

3.13

0.047

1.30

0.046

1.83

0.071

4.06

W-25

Preflow

7

5.31

0.078

3.32

0.146

1.99

0.107

3.88

w-25

Preflow

7

2.25

0.039

0.80

0.053

1.45

0.073

4.77

D-15

Preflow Postflow

9 4

2.94 2.87

0.129 0.046

1.73 1.62

0.090 0.040

1.21 1.25

0.084 0.080

4.80 4.96

D-20

Preflow Flow Postflow

7 4 3

3.50 3.41 3.33

0.115 0.056 0.173

1.79 1.70 1.56

0.094 0.051 0.113

1.71 1.71 1.77

0.083 0.087 0.075

4.06 4.06 4.20

W-A

Preflow Postflow

5 6

3.37 3.05

0.065 0.015

2.30 2.16

0.031 0.034

1.07 0.89

0.054 0.031

4.00 3.32

W-A

Postflow

5

2.69

0.084

1.89

0.123

0.80

0.091

2.87

Table

1 mM/L.

-

Mean 2 S.E.M. No.

No.

= Number

1 S.E.M.

3.42 20.20

mM/L.

1 S.E.M.

1.95 50.17

mM/L.

1 S.E.M.

1.47 fO.10

4.04 to.17

of sampks.

VI. Cerebral

glucose consumption

and glucose/oxygen

quotient Cerebral

D-5

Preflow

7

0.44

0.012

0.24

0.008

3.60

0.162

3.85

2.64

1.09

w-22

Preflow

7

0.79

0.016

0.52

0.011

4.86

0.306

4.81

4.06

0.89

W-25

Preflow

7

0.78

0.006

0.44

0.009

6.12

0.234

5.32

3.88

1.03

W-25

Preflow

7

1.11

0.028

0.86

0.028

4.50

0.144

6.62

4.77

1.03

W-A

Preflow Postflow

5 6

1.08 1.08

0.019 0.008

0.85 0.93

0.013 0.017

4.14 2.70

0.398 0.396

6.91 4.51

4.00 3.32

1.29 1.01

W-A

Postflow

5

1.18

0.039

1.00

0.045

3.24

0.342

5.18

2.87

1.35

-

0.923 +0.100

5.31 20.42

3.65 to.28

1.10 *

Mean t S.E.M.

-

No. = Number of samples. *Ninety-five per cent confidence

limits

0.691 kO.109 =

0.95

and

4.17 20.43

1.25.

per 100 grams of tissue, are comparable to those observed in the brain of adult animals. For example, the oxygen consumption and glucose utilization of the adult human brain

are 3.5 ml. and 6 mg. per 100 grams of tissue per minute, respectively.s, I1 Since the oxygen and glucose contents of the arterial blood perfusing the fetal head are considerably less

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than in the adult animal, these results are a bit surprising for they indicate the existence of a low margin of safety with respect to an adequate supply of oxygen and glucose to the fetal brain. In addition, these results are at variance with the estimates of fetal cerebral oxygen consumption made by Purves and James3 in exteriorized fetal lambs. The

Fetal

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mean cerebral oxygen consumption observed in the exteriorized preparation was approximately half the normal adult value. Obviously, more information is needed concerning the regulation of cerebral blood flow and metabolism in fetal life before the full implications of our present knowledge can be adequately appreciated.

REFERENCES

5. 6.

Amiel-Tison, C.: Biol. Neonat. 14: 234, 1969. Rudolph, A. M., and Heymann, M. A.: Circ. Res. 26: 289, 1970. Purves, M. J., and James, I. M.: Circ. Res. 24: 651, 1969. Quilligan, E. J., Hon, E. H., Anderson, G. G., and Yeh, 3.-Y.: AM. J. OBSTET. GYNECOL. 102: 716, 1968. Lucas, W., Kirschbaum, T., and Assali, N. S.: Am. J. Physiol. 210: 287, 1966. Foltz, F. M., Johnson, D. C., and Nelson, D. M.: Proc. Sot. Exp. Biol. Med. 122: 223, 1966.

Discussion J. QUILLIGAN, Los Angeles, California. The study of cerebral blood flow, oxygen consumption, and glucose utilization of the fetal lamb in utero by Makowski and co-workers is the type of investigation needed for basic understanding of how the fetus lives in, and adjusts to, his environment. The study is well designed to answer the question posed by the investigators, namely, what is the normal brain blood flow and metabolism of a fetus in utero. Davis and associates,l using the nitrous oxide method for the determination of brain blood flow in adult goats, found a value of 45 ml. per minute per 100 Gm. of brain tissue with a calculated cerebral resistance of 2.1 resistance units. This flow was slightly lower than the cerebral flows that have been reported in man2 (i.e., 54 ml. per minute per 100 Gm. of brain), while the resistance was higher (i.e., 1.6 resistance units). Although this may be a species difference, it could also occur if the arterial carbon dioxide tensions in Davis’s study were lower. Unfortunately, they did not state the carbon dioxide levels in this article. Purves and James3 noted cerebral blood flows of 48 ml. per minute per 100 Gm. of brain in their acute exteriorized fetal DR.

EDWARD

Makowski, E. L., Meschia, G., Droegemueller, W., and Battaglia, F. C.: Circ. Res. 23: 623, 1968. N. G., Schneider, J. M., Colwill, 8. Tsoulos, 1. R.. Meschia. G.. Makowski. E. L.. and iattaglia, F. C.; Pe&atr. Res. 6 182, f972. 9. Fieller, E. C.: Q. J. Pharm. Pharmacol. 17: 117, 1944. 10. Baldwin, B. A., and Bell, F. R.: J. Anat. 97: 203, 1963. 11. Kety, S. S., and Schmidt, C. F.: J. Clin. Invest. 27: 484, 1948. 7.

lamb preparation with the use of the xenon-133 technique. At a Pace, level of 40 mm. Hg, the cerebrovascular resistance was 0.82 resistance units. Makowski and colleagues in this study, using radioactive microspheres in a chronic fetal

preparation,

recorded

120

minute

ml.

per

a cerebrovascular

a cerebral per

100

resistance

Gm.

blood

flow of

of brain

and

of 0.46 resistance

units at a Pace, of 54 mm. Hg. In comparing these three states, from the adult animal to the acute exteriorized fetal lamb study to the chronic fetal preparation, it was evident that there was a gradual increase in the cerebral blood flow associated with a gradual decrease in the cerebrovascular resistance. It is well known that carbon dioxide is a potent cerebral vasodilator, and, as such, might account for the differences observed in this study as compared to that of Purves and James (i.e., Makowski and colleagues: Pace, = 54 mm. Hg; Purves and James: Pace, = 40 mm. Hg). However, in view of a recent study by Dr. Dunnihoo and myself using a chronic fetal preparation similar to that of Makowski and colleagues, it would appear that the increase in cerebral blood flow per millimeter of mercury increase in the carbon dioxide tension is less (i.e., 1.75 ml. per minute

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October 1, 1972 Am. J. Obstet. Gynecol.

et al.

per 100 Gm. of tissue per millimeter of mercury of Pace,) in the chronic state than that reported by Purves and James (i.e., 2.4 ml. per minute per 100 Gm. of tissue per millimeter of mercury of Pace,) in the acute preparation. Thus, if the cerebral blood flow in this study were readjusted from a Pace, of 54 to 40, to correspond to that reported by Purves and James, by using our value of 1.75 ml. per minute per 100 Cm. per millimeter of mercury of Pace,, then a flow of 95 ml. per minute per 100 Gm. of brain would result. This flow is still somewhat higher than was noted in the acute preparation. To further illustrate these differences between the adult animal, the acute fetal preparation, and the chronic fetal model, a comparison of the cerebral blood flow changes per millimeter of mercury increase in the carbon dioxide tension demonstrates the rather marked differences in response to carbon dioxide in these three states. The adult goat regression line was calculated by assuming that the response to carbon dioxide in the goat is of the same order of magnitude as in the adult man (i.e., 3 to 5 ml. per minute per 100 Gm. of brain per millimeter of mercury of Pace,). The acute fetal regression line value was that reported by Purves and James, while the chronic value was obtained from our data. The cerebrovascular response differences among these three states might be explained on the basis that carbon dioxide acts through a neurogenic mechanism rather than directly on the cerebral vessels. Purves and James found that the vagotomized fetus had a decreased cerebrovascular response to carbon dioxide. If the heart rate is any indicator of tonic vagal activity, then the fetus in utero has less vagotonic activity in comparison to the adult. Reynolds4 attributed this lack of vagotonia to the absence of sensory input. Certainly, the adult, as well as the acute exteriorized fetus, would have more sensory input than the in utero fetus. Unfortunately, Purves and James also noted a decrease in the cerebral gray matter flow when the vagi were sectioned, which does not fit our model. Other factors could also explain the differing response to carbon dioxide, such as the hormonal milieu of the fetus, which is quite different from that of the adult. Either estrogen or progesterone, in high levels, could blunt the fetal response to carbon dioxide. Perhaps other neurohumoral factors, such as bradykinin, might also play a role. Regardless of the etiology, the blunted response seems to be present, which makes the fetus

more dependent than the adult on the changes in cardiac output and peripheral resistance with regard to the maintenance of the cerebral blood flow. REFERENCES

1. 2.

3. 4.

Davis, L. E., Westfall, B. A., and Dale, H. E.: Am. J. Vet. Res. 25: 1159, 1963. Mountcastle, V. M.: Medical Physiology, ed. 12, St. Louis, 1968, vol. 1, The C. V. Mosby Company, p. 225. Purves, M. J., and James, I. M.: Circ. Res. 25: 651, 1969. Reynolds, S. M. R.: AM. J. OBSTET. GYNECOL. 82: 800, 1962.

DR. IRWIN H. KAISER, Bronx, New York. I would like to call attention to the developmental aspects which Dr. Makowski and colleagues and Dr. Quilligan had no opportunity to observe but which represent an opportunity, I think, to dissect a little bit further some of the variables that are of importance here. About ten years ago, Bernhard Kolmodin and I, working in Stockholm, studied the development of cortical electrical activity in the fetal sheep. As I am sure you know, it is possible to identify a period in development in which there simply is no cortical activity from an electrophysiologic standpoint. This brain is a different brain from the one at term. I don’t think that Dr. Quilligan mentioned in his discussion the duration of pregnancy of the animals he studied, but it is clear that all of the Denver group’s animals were studied at a point where the brain is fully mature, that is to say, its electrical activity is essentially the same as that of the adult brain even though the load thrust upon it by its environment may be a good deal less. Under these circumstances, I should think that it would be of immense interest to study cerebral flow, glucose utilization, oxygen uptake, and oxygen utilization in the brains of less mature animals, for example, studying the animals at 105 days rather than studying them at term. This might afford an opportunity to identify how much of glucose utilization is directed toward function and how much is directed toward growth and development of the brain. This might then offer an opening into further examination of the question of how much reserve remained for functional distress, possibly at the expense of a temporary suspension of growth. DR. W. N. SPELLACY, Miami, Florida. Some time ago, Dr. Makowski and his colleagues gave

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Fetal

insulin injections into the fetus to study glucose utilization under the influence of insulin. It is clear that the brain can shift from a glucose substrate to a ketone substrate in chronic starvation. I wonder if they have any data on the utilization of glucose or ketones by the fetal brain under conditions of hypoglycemia or maternal starvation. REFERENCE

1.

Makowski, 1970.

E. L., et al. Endocrinology

87: 710,

DR. MAKOWSKI (Closing). We agree with Dr. Quilligan that there is a substantial difference in cerebral flows in our preparation when compared with the preparation described by Purves and James and that at least part of this discrepancy can be explained by a carbon dioxide tension difference. This is one more example of the caution necessary in interpreting physiologic data on exteriorized preparations. Several group& 2 have shown other differences in physiologic measurements between the exteriorized fetus and a more intact animal preparation. Even at the high cerebral blood flow we have measured, there is a low margin of safety in terms of cerebral oxygen and glucose requirements. If one really had a cerebral blood flow of 50 ml. per 100 Gm. per minute in the sheep fetus, it would be impossible for the fetus to meet its normal cerebra1 oxygen requirement because the required arteriovenous difference of oxygen across the brain would be higher than the actual oxygen content in the arterial blood. With regard to Dr. Kaiser’s comment, it took US a fair amount of time to develop this animal

cerebral

blood

flow

and

metabolism

303

preparation. We were successful in about two thirds of the animals on whom we operated. In the remaining third, either the lateral or posterior wall of the sagittal sinus was perforated, and, consequently, the animal preparation was lost. We definitely plan to study the regional regulation of fetal brain flow in utero and attempt to correlate the magnitude of flow to the brain with its stage of growth and development. Likewise, this opens another avenue for investigation which is to study the distribution of cerebral blood flow to different areas of the brain. In this way, one could establish whether blood flow to the different areas of the brain was synchronized with the growth rate of that area or not. Dr. Spellacy brings up an interesting aspect. The studies done by Owen and co-workers3 and Smith and associates’ have shown that the brain in the human being as well as in the postnatal rat can adapt its oxidative metabolism to an alternative substrate by utilizing such keto-acids as beta hydroxybutyrate and acetoacetate. We have not thus far measured the arteriovenous difference of these keto-acids across the fetal brain in starved maternal ewes. REFERENCES

Meschia, G., Cotter, J. R., Breatnach, C. S., and Barron, D. H.: Q. J. Exp. Physiol. 50: 185, 1965. Rudolph, A. M., and Heymann, M. A.: Circ. Res., 21: 185, 1967. Owen, 0. E., Morgan, A. P., Kemp, H. G., Sullivan, J. M., Herrera, M. .G., and Cahill, G. F.. Tr.: .T. CIin. Invest. 46: 1589. 1967. Smith, “A. L., Satterthwaite, H. S.,’ and Sokoloff, L.: Pediatr. Res. 4: 379. 1970.