Acta physiol. scand. 1975. 93. 569-571 From the Brain Research Laboratory, E-blocket, University Hospital of Lund, Sweden

Rate of Energy Utilization in the Cerebral Cortex of Rats BY

BENGT NILSSON, KARIN NORBERG, CARL-HENRIK NORDSTROM and Bo K. SIESJO

There are few methods available for the measurement of metabolic rate in brain tissue and apart from a recently described technique for autoradiographic estimation of glucose phosphorylation (Reivich 1974) only one method allows regional studies. This method, which was described by Lowry et al. (1964), is based on the assumption that when the cerebral circulation is interrupted the metabolic rate is initially maintained at pre-ischemic values. Since the oxygen stores of the tissue are virtually nil, energy can only be made available from the existing stores of high-energy compounds and from glycolytic produc- tion of ATP. Accordingly, the rate of energy (-P) utilization can be calculated from the equation A - P = 2AATP + APCr -1 AADP + 2AGlucose + 2.9AGlycogen where APCr denotes the change in phosphocreatine concentration. 2.9 is a figure derived on the assumption of a 10% branching of glycogen (see Lowry et al. 1964). If glycogen is not measured, -P production from glycolysis can be obtained from changes in glucose and lactate (Gatfield et al. 1966).

One of the difficulties with the “closed box” method is that since, following decapitation, -P utilization falls continuously, the period of ischemia must be accurately assessed and the tissue must be frozen “instantaneously”. Furthermore, the validity of the method has never been tested by comparisons with other estimates of metabolic rate. The present ex- periments were undertaken to measure - P utilization in nitrous oxide and phenobarbitone anesthesia after 5, 10 and 20 s of ischemia, using rapid freezing of cortical tissue in rats. The values obtained were compared with those derived from measurements of cerebral metabolic rate for oxygen (CMR,,).

Groups of unstarved Wistar rats ( 3 U O O g) were initially anesthetized with 2-3 % halo- thane and subsequently maintained artificially ventilated on 70% N,O. Other groups were anesthetized with phenobarbitone (1 50 mg/kg). Body temperature was kept close to 37°C and arterial P,,, and Po, were adjusted to 3540 and 120-140 mm Hg, respectively. A craniectomy on one side (about 10 x 15 mm) exposed the dura over the fronto-parietal cor- tex. The tissue was frozen either without or with previous interruption of cerebral circula- tion by decapitation, using isopenthane at - 160°C which was allowed to flow across the exposed dura. The periods of ischemia, i.e. the time between decapitation and freezing of the tissue, were either 5, 10 or 20 s (N,O anesthesia) or 10 and 20 s (phenobarbitone anes- thesia). Control experiments showed that the superficial 1 mm of the cerebral cortex froze

569

570 BENGT NILSSON, KARIN NORBERG, CARL-HENRIK NORDSTROM AND BO K. SIESJO

0- N20

6-- Phcnobarbitonc

--_ ‘L - 3.0

20 --1

l o t PCr

I I

20

10 L a

1 I I Control 5 10 20

Fig. 1. Rate of changes in cerebral cortex concentrations of phosphocreatine (PCr), ATP, ADP and lactate following decapitation in rats underN,Oor phenobarbitone anaesthe- sia. Filled symbols denote significant changes from control values (p < 0.05). S.E. values not given since they were smaller than the size of the symbols.

Time of ischemia (sec)

in 1-2 s and this part of the tissue was therefore used for analyses. The tissue was dissected and extracted at - 22°C and PCr, ATP, ADP and lactate were determined using enzymatic, fluorometric techniques (see Folbergrovi et a!. 1972). Since control experiments showed that there was no significant decrease in glycogen in the periods studied -P utilization was calculated from A - P = 2AATP -(- AADP + APCr + Alactate.

Fig. 1 illustrates the changes in PCr, ATP, ADP and lactate in animals anesthetized with either N,O or phenobarbitone. The rate of fall in PCr and ATP, and the increase in lactate content, were clearly lower during phenobarbitone anesthesia. Table I shows the - P values calculated for the ischemic periods studied, and compares the values to those derived from

TABLE 1. Energy utilization in the rat cerebral cortex calculated from “closed box” and from CMRon measurements.

Anesthesia Number Time after N P utilization Energy flux (- P) of animals decapitation @mol.g-’.min-) calculated from CMRo,

(sec) Qmol. g-’. min-*)

8 5 30+2 8 10 24+2 29 4 20 17+ 1

Pheno barbitone 4 10 I l k 2 3 20 12* 1 I5

ENERGY UTILIZATION IN CEREBRAL CORTEX 571

measurements of CMR,, in nitrous oxide anesthesia (Norberg and Siesjo 1974) or in ani- mals given phenobarbitone 150 mg/kg (Nilsson and Siesjo 1975). CMR,, was converted to -P utilization by assuming that 1 pmol of glucose is completely oxidized with an energy yield of 38 pmol ATP.

The values obtained in the nitrous oxide group demonstrate that the method of Lowry et al. (1964) gives values for energy flux that are continuously decreasing within 20 s after .decapitation. However, it turns out that in these lightly anesthetized animals the - P utiliza- tion as calculated after a 5 s period of ischemia corresponds to the value derived from ,CMR,,. On the other hand, in phenobarbitone anesthesia the calculated energy flux re- mains constant and close to the measured in vivo flux at least up to 20 s of ischemia. The reason for this is not known but may signify that -P utilization is maintained close to the normal, pre-ischemic values only as long as there is a near-normal ATP concentration. Thus, the method can only be used in lightly anesthetized animals if very short ischemic periods are employed. On the whole, though, the present results demonstrate that the “’closed box” method of Lowry can be a valuable tool for studies of regional metabolic rates.

This study was supported by grants from the Swedish Medical Research Council (Projects No. 14x-2179 and 14x-263). from the Bank of Sweden Tercentenary Foundation, and by U.S. PHS Grant No. 5 R01, NS 07838-05 from NIH.

References

FOLBERGROV~, J., V. MACMILLAN and B. K. SiEsJo, The effect of moderate and marked hypercapnia upon the energy state and upon cytoplasmic NADH/NAD+ ratio of the rat brain. J. Neurochern. 1972. 19. 2497-2 505.

GATFIELD, P. D., 0. H. LOWRY, D. W. SCHULTZ and J. V. PASSONNEAN, Regional energy reserves in mouse brain and changes with ischemia and anaesthesia. J. Neurochem. 1969. 13. 185-195.

LOWRY, 0. H., J. V. PASSONNEAN, F. K. HASSELBERGER and D. W. SCHULTZ, Effect of ischemia on known substrates and cofactors of the glycolytic pathway in brain. J. bid . Chem. 1964. 239. 18-30.

NILSSON, L. and B. K. SIESJO, The effect of phenobarbitone anaesthesia on blood flow and oxygen consump- tion in the rat brain. Acra anaesthesiol. scand. 1975. In press.

NORBERG, K. and B. K. SIESJO, Quantitative measurement of blood flow and oxygen consumption in rat brain. Arta physiol. scand. 1974. 91. 154-164.

RUVICH, M., Blood flow metabolism couple in brain. In: Brain Dysfunction in Metabolic Disorders. Ed. F. Plum. Res. Publ. Ass. New Ment. Dis. Raven Press New York. 1974. 53.