Influence of early chronic phenobarbital treatment on cerebral arteriovenous differences of glucose and ketone bodies in the developing rat

ht. I. Devl. Neuroscience, Printed

Vol. 9,

No. 5, pp. 45M61,

1991.

0736-5748/91

$3.00+0.00

Pergamon Press plc 0 1991ISDN

in Great Britain.

INFLUENCE OF EARLY CHRONIC PHENOBARBITAL TREATMENT ON CEREBRAL ARTERIOVENOUS DIFFERENCES OF GLUCOSE AND KETONE BODIES IN THE DEVELOPING RAT HENRI SCHROEDER,* LAURENT BOMONT and ASTRID NEHLIG INSERM U272, Universit& de Nancy I, 30 rue Lionnois, B.P. 3069.54013 Nancy Cddex - France (Received

27 August 1990; in revised form 7 January 1991; accepted

14 January 1991)

Abstract-The

influence of an early chronic phenobarbital treatment on cerebral arteriovenous differences of glucose, lactate, pyruvate, P-hydroxybutyrate and acetoacetate was studied in suckling rats. The animals were treated from day 2 to 21 after birth by a daily injection of 50 mg/kg phenobarbital or by saline and were studied at 10, 14 and 21 days. Phenobarbital treatment induced a decrease in cerebral arteriovenous difference of glucose at P14 and no change at PlO and P21. The barbiturate did not have any influence on cerebral arteriovenous difference of lactate and pyruvate at the three stages studied. Cerebral uptake of P-hydroxybutyrate was unchanged at PlO and increased by two-fold at P14 and by threefold at P21 by phenobarbital. Cerebral arteriovenous difference of acetoacetate was low and did not change with the pha~a~ologica~ treatment. At P14 and P21, the calculated amount of oxygen used by the brain for the oxidation of ketone bodies was twice as high in barbiturate- as in saline-treated rats and reached values of 47 and 16% respectively in phenobarbital-exposed animals. In addition, the barbiturate seemed to affect the carrier process of P-hydroxybutyrate from blood to brain. The results of the present study are in good agreement with previous data from our laboratory showing that an early chronic phenob~bital treatment is able to induce a shift in the cerebral energy metabolism balance in favor of ketone bodies. Keywords: glucose, lactate, P-hydroxybutyrate, cerebral metabolism, postnatal phenobarbital, barbiturate, chronic treatment, cerebral arteriovenous differences.

development,

It has been known for almost two decades that, as a result of the high lipid content of maternal milk,” the infant rat develops a marked ketosis within a few hours following birth” and ketone bodies, i.e. acetoacetate (ACA) and ~-hydroxybutyrate (PHB) are actively taken up and utilized by the immature brain *’ . Indeed ketone bodies appear to be substrates more efficient than glucose both for amino acid,‘,” an& lipid biosynthesis3’,49 m * the developing rat brain. In addition, the activities of the enzymes regulating ketone body metabolism reach high levels in the suckling rat brain. 1,3* In a previous work from our laboratory, we showed that a chronic neonatal treatment from day 2 through 35 with the commonly used anticonvulsant drug phenobarbital (PhB) was able to induce a shift in the cerebral energy metabolism balance. This shift seemed to be related to decreased circulating levels of glucose and increased blood concentrations of both PHB and ACA. It translated into a decrease in the rate of biosynthesis of cerebral amino acids from [2-‘4C]gIucose accompanied by a simultaneous rise in the efficiency of [3-14CjpHB as a precursor for these same amino acids, without any marked change in the final concentration of cerebral amino acids at the different stages studied.33*34 It is known that ketone bodies are taken up at a rate which is proportional to their arterial concentration6S16*20and this relationship between circulating levels and cerebral uptake of ketone bodies persists in situations such as starvation, 6*20Thus, to determine the importance of blood concentrations as a factor in the regulation. of cerebral ketone body utilization in a pharmacological situation such as PhB exposure and also to determine whether the changes in the relative rates of utilization of glucose and PHB induced by PhB in the immature rat brain are correlated with modifications in the rates of uptake of these substrates by the brain, in the present study we measured the cerebral arteriovenous differences of glucose, lactate, pyruvate and ketone bodies in control and PhB-treated rats during the suckling period. *Author to whom offprint requests should be addressed. ACA, acetoacetate; PHB, P-hydroxybutyrate;

Abbreviations:

453

PhB, phenobarbital.

H. Schroeder et al.

454

EXPERIMENTAL

PROCEDURES

Animals and pharmaco~5~ica~ treatment

Two adult female Sprague-Dawley rats and one male were mated together for 7 days. The animals were maintained on a 12: 12 h light dark cycle (lights on at 0690 h). Food and water were available ad libitum. After delivery, litter size was reduced to 12 pups for homogeneity. Rat pups received a daily subcutaneous administration of PhB at the dose of 50 mg/kg from day 2 to 21 (day of birth was considered as day 0). Control animals received an equivalent volume of saline. The pharmacological treatment was the same inside each litter, either saline or PhB for all animals to get pups all about the same size in the litter and to prevent the mother from rejecting the lowweighted PhB-treated rats. However, to avoid a litter effect, the experiments were performed on three different litters at least for each treatment and each stage studied. Daily injections were performed between O&O0and 0830 a.m. and the experiments were performed about 3 h later, i.e. between lo:30 and 11:30 a.m. The animals were studied at the postnatal age of 10 (PlO), 14 (P14) and 21 days (P21). Collection of blood samples

For the measurement of blood levels of glucose and ketone bodies, the rats were killed by decapitation and blood was collected in a prehepa~nized glass dish. For the dete~ination of cerebral arteriovenous differences, rats were anesthetized by intraperitoneal injection of an equithesine solution at the dose of 0.3 ml/l00 g body weight. Sinus blood (100 to 250 p,l according to the age of the animal) was first taken by insertion of a 25 gauge needle into the confluence of the sinuses. Arterial blood was sampled immediately after from the femoral artery, which had been previously dissected out and separated from the femoral vein and sciatic nerve. Arterial blood was obtained after section of the femoral artery and collection of the blood was performed into heparinized 60 ~1 glass capillary tubes. The volume of arterial blood sampled ranged from 60 to 240 l.~las a function of the age of the animal. Treatment

of the

blood samples and analytical methods

For glucose, blood samples were immediately centrifuged and arterial and venous plasma glucose levels were measured on 10 l~,lsamples in a Beckman glucose analyzer. For all other measurements, blood samples were immediately deproteinized with 10% perchloric acid and centrifuged at 3000 ‘pm at 4°C. The supematant solutions were neutralized by 20% KOH and used for the spectrophotometric enzymatic determination of ACA3*, PHB”, lactate and pyruvate.20 Statistical analysis

Data of the PhB-treated animals were compared with those of the control group by means of a Student’s t-test. The postnatal evolution of arterial and venous blood levels as well as that of cerebral arteriovenous differences of glucose, lactate, pyruvate and ketone bodies were analyzed by comparing the data at one stage with those at the preceding stage by means of Bonferroni multiple comparison procedures. I5 RESULTS Circulating blood leve&

of glucose

and ketone bodies

Blood levels of glucose were si~ific~tly higher in saline- than in PhB-treated rats, by 14% at PlO and by 6 to 7% at P14 and P21 (Fig. 1). Blood glucose concentrations did not change in any group over the period of development studied. In control rats, blood levels of l3HB and ACA reached their highest values by PlO and P14 with no change between these two stages and significantly decreased by 73 and 58% respectively between P14 and P21. In PhB-treated rats, postnatal evolution of ACA blood concentrations was similar to the one in control rats, whereas l3HB blood levels reached high values at PlO but significantly increased by 52% to reach a very high peak at P14 and then decreased by 77% between P14 and P21. At all stages studied, circulating ketone

Phenobarbital,

455

glucose and ketone body uptake

GLUCOSE

ACA

MS *

c5 1

E0

a-

2.0.

m

5.

1.5.

5

4-

1.0.

Q

2.

0.5.

i

OA

iii

O_

PlOPl4ml

PlOPl4

P2l

PlOPuP

Fig. 1. Influence of early PhB treatment on circulating levels of glucose, 8-hydroxybutyrate (BHB) and acetoacetate (ACA) in the developing rat. *P
body levels were significantly higher by 27 to 99% in PhB- than in saline-treated difference was especially marked at P14 for 8HB (Fig. 1).

animals and this

Arterial and venous blood levels and cerebral arteriovenous differences of glucose, lactate and pyruvate

Arterial and venous blood glucose concentrations were higher in saline- than in PhB-treated rats at P21, but they were similar at PlO and P14 in both groups of animals (Table 1). Cerebral arteriovenous difference of glucose was lower in PhB- than in saline-treated rats at P14 and similar in both groups at PlO and P21. Arterial and venous blood concentrations of lactate did not change over postnatal development both in saline- and PhB-treated animals. However, the barbiturate treatment induced a significant decrease in arterial and venous blood levels of lactate at P14 and P21, as compared to control values. The cerebral arteriovenous difference for lactate was the same in both groups of animals at the three stages studied. It decreased in saline- and PhB-exposed rats by about 30% between PlO and P14 but this change was not significant. Arterial and venous blood levels as well as cerebral arteriovenous differences of pyruvate were about 8 to 28 times lower than those of lactate. However, there was no change over the postnatal period studied and no significant difference between PhB- and saline-treated rats for these three blood parameters studied (Table 2). Arterial and venous blood levels and cerebral arteriovenous differences of P-hydroxybutyrate and acetoacetate

Arterial and venous blood concentrations of 8HB were 2 to 3 times higher at P14 than at PlO in both groups of animals. They then decreased by about 5 times in saline- and by about 2 times in Table 1. Influence of phenobarbital (PhB) on arterial (A) and venous (V) concentrations and cerebra1 arteriovenous differences (AVD) of glucose in developing rats Stage PlO PI4 P21

A Control PhB Control PhB Control PhB

10.0~0.1 9.620.2 10.8 2 0.2 10.9kO.4 * 12.5 k 0.5” 11.4kO.3”

V 8920.1 8.3 + 0.2 9.7kO.3 10.2 f 0.4** 11.4*0.4** 10.3 f 0.3’

AVD +1.2*0.1 +1.3*0.1 +1.1 kO.1 +0.8 kO.1”’ +1.1 kO.2 +1.1 -co.1

Values, expressed as mmol/l, are means 2 S.E.M. of 9 to 18 animals. ‘P
H. Schroeder et al.

456 Table

2. Influence

of phenobarbital (PhB) on arterial (A) and venous differences (AVD) of lactate and pyruvate

(V) concentrations in developing - rats

I.actate Stage PlO P14 P21

Control PhB Control PhB Control PhB

A

V

AVD

A

2.97 _f 0.52 2.46 t 0.34 3.21 t 0.25 2.44 + 0.24” 3.25-t0.26 2.04 t 0.29

4.06 t 0.75 3.47 to.51 3.92-to.31 3.12kO.29’ 4.05 -c 0.33 2.91 + 0.28h

-1.09i-0.30 - 1.01 + 0.25 -0.71 ? 0.12 --0.68kO.10 --0.80t0.12 -~0.87?0.19

0.14t0.01 0.16r0.01 0.15t0.01 0.15~0.01 0.16 + 0.01 0.17 t 0.01

between

\’

control

PI0 P14 P21

0.19 0.20 0.20 O.lY 0.19 0.20

t 0.01 + 0.01 k 0.01 Iro.01 ? 0.01 If-0.01

AVD -0.05 1rc_ 0.011 -0.043 2 0.006 0.008 -0.051+ -0.041 -r 0.005 -0.031 ?I 0.004 -0.033 t 0.007

and PhB-treated

rats at Clgtvcn

of phenobarbital (PhB) on arterial (A) and venous (V) concentrations and on cerebral differences (AVD) of P-hydroxybutyrate and acetoacetate in developing rats P-Hydroxybutyrate

Stage

A Control

0.84-tO.15

PhB Control PhB Control PhB

0.92kO.10 1.72&0.21** 1.73+0.22** 0.36?0.09** 0.76 k 0.09b**

V 0.51~0.11 0.61’0.10 1.50-t0.16** 1.28?0.21*’ 0.3OirO.O8** 0.60+0.09”**

arteriovenous

Pyruvate

Values, expressed as mmol/l, are means rt S.E.M. of 7 to 17 animals. a:P
and cerebral

arteriovenous

Acetoacetate AVD +0.33 k 0.05 +0.31 2 0.03 +0.22 t 0.05 +0.45 f 0.03’** +0.06 t 0.01 +0.16t0.02’**

A 0.17~0.01 0.25 t 0.03h 0.18 + 0.03 0.25 t 0.03 0.10t0.01** 0.16 +- 0.02”

V 0.13”0.02 0.19 2 0.02” 0.1310.03 0.22 k 0.03 0.08 k 0.01 0.14 t 0.02h

Values, expressed as mmol/l, are means 2 S.E.M. of 4 to I1 animals. *P
AVD +0.038 +0.061 +0.048 +0.027 +0.022 +0.019

+ ” k lr 2 t

0.006 0.015 0.016 0.008’ 0.005 0.002

or PhB-treated rats at a given

PhB-treated rats between P14 and P21. The cerebral arteriovenous difference for PHB regularly decreased in control rats throughout the period studied, whereas it followed the same evolution as blood levels, reaching peak levels at P14 in PhB-treated rats (Table 3). Arterial and venous blood levels of PHB were similar in both groups of animals at PlO and P14 and twice as high in PhB- as in saline-treated rats at P21. Cerebral arteriovenous differences for /3HB were similar in both groups of animals at PlO, and 2 to 3 times higher in PhB- than in saline-treated rats at P14 and P21. Arterial and venous blood ACA concentrations and cerebral arteriovenous differences of ACA reached their highest levels at PlO and P14 and decreased by 36 to 118% between P14 and P21 (Table 3). Circulating levels of ACA were higher in PhB- than in saline-treated rats at the three stages studied. Cerebral arteriovenous differences for ACA were about 3 to 15 times lower than those for PHB. They slightly rose between PlO and P14 and decreased between P14 and P21 in control rats. In PhB-treated animals, they were highest at PlO and markedly decreased between PlO and P14. Calculated oxygen equivalents

The percentage of calculated oxygen equivalents used for the oxidation of glucose reached 70 to 80% at PlO and P14 in control animals and increased to 92% at P21 (Fig. 2). In PhB-treated rats, this value was about 70% at PlO, decreased to 53% at P14 and rose again to reach 84% at P21. In saline-treated animals, the percentage of calculated oxygen equivalents used for the oxidation of ketone bodies (PHB plus ACA) decreased from the value of about 30% at PlO to 8% at P21. In PhB-treated rats, this value was 20% at PlO, increased to reach the level of 47% at P14 and then dropped to 16% at P21. Correlations between arterial concentration and cerebral arteriovenous difference of (5hydroxybutyrate

As shown in Fig. 3, there was a significant linear correlation between arterial concentration and cerebral arteriovenous difference of PHB in control animals at the three stages studied.

glucose and ketone body uptake

Phenobarbital,

457

100.

0

l-l

Control

80.

00.

40.

20.

Oh

QLU

KB

ULU

KB

PlO

(1LU

P14

KB

P21

Fig. 2. Influence of early PhB treatment on the percentage of calculated oxygen equivalents used for the oxidation of glucose and ketone bodies in the developing rat brain. The amount of oxygen necessary for glucose oxidation was calculated by using the formula [glucose-(lactate+pyruvate)/2] x 6, and for ketone bodies by using [@HB X 45)+(ACA X 4)]. Abbreviations: GLU, glucose; KB, ketone bodies. % Control -% PhB 0.6

PlO

0.4

‘, --l-

0.2

0 0 0 ~.O.ODI*o.IaOx r-o.,, ~,~O.OO _I??______-----

0..

y.O.le~r0.0011 1.0.03

0IL_

A 5 g

0.6 0.4

6

1

3

1_/1_ ~~0.41~*0.02~1

P14

_____--w----

. r.0.11 _!O__ -0

0.2

0

o

e 4

2

0

1

O

p-o., o

o

40*0.2

r.O.BT

0

2

II

I

(P80.01)

3

y.-0.040+0.3~~r

r.o.00

A

(P*o.oI)

pHB (mmol/l)

Fig. 3. Influence of early PhB treatment on the correlation between arterial concentrations (A) and cerebral arteriovenous differences (AVD) of g-hydroxybutyrate (gHB) in developing rats. The values are expressed as mmoM.

Conversely, in PhB-exposed rats, there was no significant correlation between arterial level and cerebral arteriovenous difference of PHB at any stage studied. In addition, the slopes of the regression lines were statistically significantly different in saline- and PhB-treated rats at the three stages studied (P
458

H. Schroeder

et al.

DISCUSSION In the present study, we confirmed that, in control animals, cerebral uptake of glucose does not change over the period of development studied, as previously shown in rats,” human infants’“.a’.4’ and chickens.25 Lactate release by the brain decreases by about 30% between PlO and P14, however this change is not significant. This decrease in lactate release is not likely to be correlated with an increase in the rate of glycolysis in the brain. Indeed, local cerebra1 metabolic rates for glucose (LCMRglc) are very low both at PlO and P14 in the rat and only represent about 35% of the adult 1eve1s.26In addition, the activity of glycolytic enzymes stays low until P14 and markedly increases between P14 and P21,‘.21 paralleled by a sharp rise in the levels of cerebral glucose uti1ization26 but there is no further change in the amount of lactate released by the brain of control rats between P14 and P21 (Table 2), which is in good agreement with the increased rate of glucose utilization. Indeed, it is known that the part of the glucose taken up and not used by the brain is released as lactate and pyruvate. 5,6.16In addition, Cremer and Heaths showed that, in the l&day-old rat, glucose does to represent the major source of lactate, which is mostly originating in the blood. Indeed, the blood-brain barrier is much more permeable to lactate and pyruvate in the 15-l&day-old rat.6 So, the major part of lactate released by the immature brain would originate in a blood pool and the increase in the cerebral arteriovenous difference of lactate between PlO and P14 may be partly the reflection of the two-fold increase in lactate arterial concentration over that period (Table 2). The quite high lactate release from brain to blood is also known to be the result of the active utilization of ketone bodies,‘6,29,3” which occurs both in PhB- and salinetreated rats in the present study. Pyruvate release by the brain remains low and mostly unchanged between PlO and P21, which is usually the case in most situations according to its fast metabolism and low blood concentration, as previously shown. “J”,~“,~’ In control rats, PHB and ACA cerebra1 uptake is highest at PlO and P14 and largely decreases at P21 (Table 3). Ketone bodies represent about 20 to 30% of the oxygen used by the brain at PlO and P14 and only 8% at P21 (Fig. 2) which is in agreement with previous data16,‘V,2s.35.40.41 showing that ketone bodies act as alternate substrates in the suckling rat brain and represent as much as 30% of the cerebra1 energy metabolism balance. Cerebra1 uptake of ketone bodies has been shown to be proportional to their circulating levels during postnatal deve10pment,1”~19,2”which is true in the present study in 14- and 21-day-old control rats. However, at PlO, PHB arterial concentration is twice as low as at P14 whereas cerebra1 arteriovenous difference is by 50% higher at PlO than at P14. There are no data available in the literature at so early stages in the rat, but this result seems to agree with the data of Kraus et ~1.‘~ who showed that the brains of human newborns and infants are able to remove and oxidize ketone bodies at rates 4 to 5 times higher than adults at a given arterial concentration. The influence of an early chronic PhB treatment on the postnatal evolution of cerebral arteriovenous differences of glucose and ketone bodies has not been studied previously. It has been shown that barbiturates administered at anesthetic doses depress cerebra1 glucose metabo1ism2~3~2s~3” mainly by inhibiting a key enzyme of glycolysis, phosphofructokinase.-‘,” In addition, barbiturates, given at anesthetic doses, are able to modify rate constants for glucose and deoxyglucose transport into the brain”,j6 and to reduce cerebral blood flow and cerebra1 oxygen consumption. 1s.28In the present study, the effects of an early chronic PhB treatment at anticonvulsant doses on glucose uptake by the brain vary according to the stage studied. At PlO, cerebral glucose uptake, lactate and pyruvate release are similar in PhB- and saline-treated rats and the calculated amount of oxygen used by the brain for glucose oxidation reaches a similar value of 80% in lo-day-old saline- and PhB-exposed animals. By contrast, at P14, cerebra1 glucose uptake is reduced by 32% in PhB- as compared to saline-treated rats whereas lactate release is similar in both groups of animals. At that stage, the calculated amount of oxygen used by the brain for glucose oxidation is also reduced by 32% in PhB-exposed animals as compared to controls. At P21, cerebra1 glucose uptake and lactate release reach the same levels in both groups of animals. The data at P14 and P21 are in good agreement with our previous findings on the effect of a similar PhB treatment on the rate of glucose utilization for cerebral amino acid biosynthesis.“3 Indeed, PhB treatment is able to induce a decrease in the efficiency of this metabolic pathway at P14 but no change at P21. The lack of effect of the chronic anticonvulsant barbiturate treatment

Phenobarbital, glucose and ketone body uptake

459

both on cerebral glucose uptake and amino acid biosynthesis at P21 may originate in the decrease of the circulating level of PhB, which reaches the values of 70,SO and 53 FmoVml at 3 hrs after its administration at PlO, P14 and P21 respectively,33 as well as in a possible tolerance phenomenon to the drug and/or in the more active clearance and shorter half-life of the drug at that stage, as previously shown in rodents.“*39q46,47 Cerebral uptake of PHB is the same at PlO in PhB- and saline-treated rats, whereas at P14 and P21, barbiturate exposure induces a two- to three-fold increase in the rate of cerebral uptake of PHB over control values. In parallel, the calculated amount of oxygen utilized by the brain for ketone body oxidation is similar in both groups of animals at PlO and about twice as high in 14and 21-day-old PhB-exposed as in control rats. These results are in good agreement with our previous data showing that an early chronic PhB treatment is able to induce an increase in the rate of utilization of l3HB as a precursor for cerebral amino acid biosynthesis in the suckling rat.” Since cerebral l3HB uptake has been shown to be proportional to its circulating level,” the increase in cerebral uptake and utilization of l3HB is likely to be related to the rise in the circulating level of this ketone body induced by PhB, especially marked at P14 (Fig. 1). The rise in ketone body circulating levels may in turn originate in the increase in the activity of the enzymes catalyzing the degradation of fatty acid in the liver, since PhB is well known to be an effective inducer of hepatic microsomal enzymes. t7S43The increase in cerebral BHB utilization may also be related to an induction of ketone body metabolizing enzymes in the brain. Indeed, after a five days PhB treatment in the adult rat, the activity of gamma-glutamyltransferase is increased by 50% in cerebral microvessels and this induction lasts at least for two weeks after the end of the treatment.14 In addition, the increase in cerebral arteriovenous difference of PHB may also originate in changes in the transport of this ketone body from blood to brain. In control humans and animals, ketone bodies are taken up by the brain at a rate which is proportional to their circulating levels. t6,t9~20 However, in PhB-treated rats, on the opposite to control animals, there is no significant correlation between the arterial concentration and the cerebral arteriovenous difference of PHB (Fig. 3). In addition, the slopes of the regression lines for PHB are significantly different between the two groups of animals at the three stages studied. It then appears that PhB seems to be able to change the properties of the transport system for ketone bodies at the level of the blood-brain barrier. As a consequence, whereas in control rats, l3HB and ACA transport represents a site of regulation of ketone body utilization by the brain8 this may not be true any longer in PhB-treated rats. The impairment in l3HB transport system at the level of the bloodbrain barrier may be partly the reason why the effects of PhB on PHB uptake last until P21 (Table 3), whereas barbiturate treatment does not impair glucose uptake after P14 (Table 1). In addition, since lactate and BHB are transported from blood to brain by the same carrier process,’ it appears also possible that PhB could disturb the release of lactate as well as the uptake of PHB by the brain. Because of the sedative effects of PhB, it can be questioned whether undernutrition and reduced caloric intake may accompany barbiturate administration in rat pups. However, in the present study, we could not notice any si~ificant difference in the frequency of suckling of PhBtreated rat pups, as compared to controls, except for about one hour after the barbiturate administration and only during the first few days of life. In a previous study on artificially reared rat pups, using chronic intragastric feeding canulas, Diaz et al.” could demonstrate that PhB retards brain growth of the infant rat without affecting body growth and that this effect is not undernut~tion-mediated. Moreover, the neuromo~hological deficits induced by a chronic PhB treatment, especially at the level of the dendritic tree of cerebellar Purkinje cells are very mild* in contrast to the extensive damage induced at this level by undernutrition.37 In fact, PhB-induced effects on the central nervous system are rather similar to deficits observed after ethanol treatment. Ethanol and barbiturates are both sedative hypnotics which cause similar depression of the depolarization induced by calcium influx across synaptosomal membranes in adult animals.*3.22 They both have a comparable neurotoxic effect on the developing brain and unlike many other drugs or insults, they both destroy not only proliferating, migrating and differentiating cells, but even already formed neurons. In addition, the types of cell and layers affected, and the extent of the deficits appear to be very similar with both agents.45 It then appears from these data as well as from the very moderate body weight deficits of PhB-exposed rats as compared to controls that the ON9:5-B

460

H. Schroeder

et al.

effects of PhB observed in the present study are likely to be mainly direct effects, not undernutrition-mediated. In conclusion, the results of the present study show that an early chronic PhB treatment is able to induce an increase in the rate of cerebral uptake of PHB at P14 and P21 and a decrease in cerebral arteriovenous difference of glucose at P14. At PlO and P14, glucose represents 70 to 80% and ketone bodies 20 to 30% of the total amount of oxygen utilized by the brain of control rats and these values shift to 92 and 8% respectively at P21. However, in PhB-treated rats, especially at P14 and P21, ketone bodies are more efficiently used by the brain and represent about 4.5and 16% respectively of the total amount of cerebral oxygen utilization. These results are in good accordance with our previous findings, showing that PhB is able to induce a shift in the cerebral energy metabolism balance, translating into a decrease in the rate of cerebral amino acid biosynthesis from glucose and an increase in the efficiency of PHB as a precursor for cerebral amino acid biosynthesis. 33,34This shift in cerebral energy metabolism balance protects the brain against a depletion in amino acid concentration. However, it also apparently maintains the brain in a prolonged period of immaturity, translating into a longer dependency upon ketone bodies as substrates for energy metabolism and biosyntheses, which may in turn, as we showed in previous studies, induce some impairment in learning abilities of adult rats exposed to phenobarbital in the early postnatal period.” Acknowledgements-The

authors wish to express their gratitude to Prof. Paul Vert, Director of INSERM U272 for his encouragement, advice and support. This work was supported by the Institut National de la Sante et de la Recherche Medicale (U272) and by a grant from the Fondation pour la Recherche Mtdicale. The editorial assistance of L. Gouret is gratefully acknowledged.

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