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Energy Metabolism: Valid Techniques in Humans and Animals
[9]
Cerebral Glucose/Energy Metabolism" Valid Techniques in Humans and Animals Siegfried Hoyer
Introduction The healthy, mature, nonstarved mammalian brain uses glucose only by oxidizing it to obtain energy in the form of ATP, which is necessary to maintain cellular function. A study of the metabolic pathway of glucose and the energy pool in the brain is of interest in evaluating both normal function and pathological states. The normal supply of the substrates oxygen and glucose to the brain is guaranteed by the blood flow in the carotid and vertebral arteries. Cerebral blood flow (CBF) and cerebral oxidative metabolism have been found to be tightly linked functionally. Therefore, it seems necessary also to touch on the control mechanisms of CBF. The mammalian brain is a multiorgan organ; that is to say, it has a heterogeneous structure and consists of functionally different regions. This holds true for the gray matter in the cerebral cortex and for the diverse subcortical nuclei. Cerebral gray and white matter have to be regarded as distinct from one another in functional terms. It is as yet not known whether the white matter is also characterized by a regional diversity similar, in metabolic terms, to that of the gray matter.
Control Mechanisms of Cerebral Blood Flow There are two physiological parameters that control CBF: (1) the mean arterial blood pressure (MABP) and the cerebral perfusion pressure (CPP) and (2) the partition pressure of carbon dioxide in arterial blood (paCO2). Under normal conditions, MABP is around 100 mmHg in humans and in mammals (monkey, dog, cat, rat) commonly used for experimental purposes. Normal CBF is maintained over an MABP range of around 50 to around 150 mmHg (autoregulation of CBF). However, the cerebral metabolic rates of glucose, lactate, and CO2 change under moderate arterial hypotension (1). The relationship between MABP and CBF is valid only when the intracranial pressure (ICP) is normal (close to zero). When ICP is increased as a result 124
Methods in Neurosciences, Volume 30 Copyright 9 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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of brain edema or other space-occupying lesions, the relationship between CPP and CBF becomes valid (CPP = MABP - ICP) (2). The physiological, normocapnic paCO2 is around 40 mmHg. Hypocapnia induces a fall and hypercapnia induces an increase in CBF in a rather linear manner between paCO 2 of around 20 and 80 mmHg. In this range, the cerebral metabolic rates of oxygen, CO2, glucose, and lactate are kept constant by an increase in the extraction rates of the substrates and vice versa (3).
Physiological Steady State In studies designed to investigate parameters of the cerebral glucose and oxidative energy metabolism, MABP and paCO 2 have to be maintained in physiological ranges. MABP can be monitored either by means ofa sphygmomanometer (preferred method for humans) or via an arterial catheter and pressure transducers (preferred method for experimental animals). Although the arterial partition pressures of oxygen (paO2) do not control CBF, a fall from normoxemic (around 100 mmHg) to hypoxemic values (around 80 mmHg or lower) has been shown to induce changes in cerebral glucose/energy metabolism (4-6). Another parameter that has to be kept normal is the arterial glucose concentration. Arterial hypoglycemia induces a more marked reduction in cerebral glucose consumption than in oxygen utilization (7), and drastic changes in energy metabolism in the cerebral cortex (8). Besides the arterial blood constituents pCO2, PO2, and glucose, further parameters are of importance because of their capability to vary CBF and thus, secondarily, cerebral oxidative metabolism: hematocrit, hemoglobin, and pH. To complete the steady-state parameters, body temperature (37~ has to be recorded either rectally (humans, experimental animals) or intraperitoneally (experimental animals). It is generally accepted that the maintenance of the above steady-state condition over an experimental period of at least 15 minutes guarantees the comparability of different studies. However, in human disease states, or in defined pathophysiological experimental conditions, both quality and quantity of the variations in the respective steady-state parameter will have to be strictly maintained.
Studies in Humans Various techniques are available for the investigation of global and local cerebral utilization rates of oxygen and glucose.
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Kety-Schmidt Technique Global measurements of substrate consumption of any organ are based on the Fick principle: the amount of a substrate that is used by that organ is represented by the difference in the concentrations measured in the arterial blood being supplied to it and in the venous blood released from its, i.e., the arteriovenous difference. However, there are conditions that have to be fulfilled for this principle to hold: the arterial and venous concentrations of the substrates and the blood flow through the organ have to be constant during the period of measurement (steady-state condition; see above), and one main vein only should drain the blood from the organ. The consumption or release of a substrate can then be calculated from the arteriovenous substrate difference and blood flow (9). It can be assumed that any substrate concentration is identical in the blood of all larger arterial vessels of the body. Therefore, there is no need to sample arterial blood from the carotid artery; it can be more conveniently sampled from the femoral artery, for example. The main venous drainage from the brain takes place via the internal jugular vein. However, this vein is joined by the facial, lingual, pharyngeal, and thyroid veins, so that the extracerebral contamination is considerable. Proximal to these veins, the superior bulb of the internal jugular vein is situated in the posterior part of the jugular foramen. Mixed cerebral venous blood, i.e., blood from both cerebral hemispheres, the brain stem, and the cerebellum, with only minor extracerebral contamination, can be collected from the superior bulb of the internal jugular vein by direct puncture. A modification (10) of the original Kety-Schmidt technique (9) facilitates blood sampling in that integral concentrations of blood substrates are collected over 10 minutes by motor syringes extracting 1 ml/ minute of blood, to avoid extracerebral contamination at the venous site in particular. With respect to the measurement of global CBF it must be borne in mind that the tracer used to calculate blood flow must be nontoxic, must not produce any side effects, must not be varied metabolically or itself induce variations in metabolism, should be insoluble or only slightly soluble in blood and adipose tissue, should diffuse rapidly into the brain, should be easily analyzed, should have a known brain-blood partition coefficient, and should be economical in use. Some candidates largely fulfill these prerequisites" nitrous oxide, argon, and both labeled krypton and xenon. Nitrous oxide in low concentration (15-20% in air) is most frequently used, although the calculation of CBF from a 10-minute saturation period is overestimated by 10-15%. This error can be minimized by prolonging the saturation period to 14 minutes and extrapolating to infinity (11). Although this Kety-Schmidt technique, which allows global measurements only, has some limitations, it
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has the advantage of providing data on both CBF and the utilization rates of substrates of interest. Several very sensitive analytical techniques are available for the measurement of cerebral arteriovenous substrate differences, such as oxygen and CO2 (gas chromatography), glucose, lactate, pyruvate, and ketone bodies (spectrophotometry), amino acids (high-performance liquid chromatography), free fatty acids (gas chromatography), etc. Uptake or release of these substrates and the shifts in the pattern of different metabolic pathways can be followed. Furthermore, the duration of one study is around 30-40 minutes from the puncture of the vessels to removal of the needles. It may be easier to maintain a steady state over this period than throughout longer lasting investigations (see following), which makes this method more practical for use in mentally deranged people. Although this technique is invasive, it can be applied almost daily if necessary without causing excessive stress to the patient. Finally, the costs are quite low.
Positron E m i s s i o n T o m o g r a p h y T e c h n i q u e s Positron emission tomography (PET) techniques are based on a modified Fick principle (see previous) in which radioactive tracers are used to determine the cerebral utilization rates of substrates. The direct measurement of radioactivity in the brain tissue replaces the determination of the substrate concentration in the mixed venous blood of the superior bulb of the internal jugular vein. Techniques are developed to investigate the utilization rates of glucose and oxygen. Tracers used are 18F-labeled a-fluoro-deoxyglucose (FDG), [llC]methyl-D-glucose, and 150, The FDG method is the one most commonly used in studies of regional glucose utilization by means of PET. This method is based on the principles outlined by Sokoloff et al. (12) and Reivich et al. (13). Deoxyglucose uses the same carrier at the blood-brain barrier for transport from arterial blood into the brain tissue. Here it is phosphorylated by hexokinase but not converted further into fructose 6-phosphate. Deoxyglucose 6-phosphate is trapped in the brain, and its resulting accumulation is the marker to be quantified by PET. Deoxyglucose 6-phosphate is not a substrate for glucose6-phosphate dehydrogenase, but can be hydrolyzed by glucose-6-phosphatase. For calculation of the local metabolic rate of glucose, the time interval between injection and PET measurement, the plasma concentrations of glucose and FDG during the study, the rate constants of the transmembranous carrier system, of the FDG phosphorylation step and the "lumped constant" must all be known. For studies under normal conditions, the rate constants and the lumped constant can be derived from animal experiments. However,
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under pathologic conditions, individual determination of the rate constants by dynamic PET scanning is thought to be more accurate (14, 15). The determination of local cerebral oxygen consumption is more crucial because of the very short half-life of oxygen-15, which is 2.1 minutes. Oxygen- 15 is inhaled and is almost entirely metabolized to labeled water (H2150). Three emission scans have to be carried out during the inhalation period for quantitative measurement of the metabolic rate of oxygen (16, 17). The immobility of the subject's head is a crucial factor for any PET study. Because the duration of one investigation either of local glucose utilization or of local oxygen consumption is around 1 hour, maintenance of the steady state may become difficult, especially because the patients concerned are often (mentally) restless and uncooperative (demented). Clearly, the PET technique can yield very precise information on regional oxidative metabolism and thus on pathobiochemical pathways in diseased states. Otherwise, an unequivocal disadvantage lies in the high costs (cyclotron, PET) and in the availability of highly trained specialists. Therefore, the availability of such equipment is restricted to a few research centers.
Nuclear Magnetic Resonance In recent years, high-resolution nuclear magnetic resonance (NMR) spectroscopy has been established as a noninvasive technique to investigate phosphorus resonances from brain tissue (18, 19). However, technical difficulties and instrumental limitations still restrict the wide application of NMR spectroscopy. Experimental data so far available on normal and diseased states of cerebral metabolism are characterized by considerable scatter.
Postmortem Studies Postmortem studies of cerebral glucose/energy metabolism are limited to compounds that do not vary at all, or hardly at all, during ischemia/anoxia of the brain in the agonal state. This precludes the reproducible investigation of labile phosphates. Apart from these, enzyme activities of the glycolytic and oxidative glucose breakdown processes (see following) can be validly studied during the first 2 days after death. However, interpretation of the results has to be adapted to allow for the postmortem changes occurring as a result of room temperature and the time lapse before tissue sampling after death. To this end, relevant animal experiments have to be performed to test how and to what extent room temperature and temperature in the cool
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chamber, both for different durations, affect the activities of the enzymes to be studied.
S t u d i e s in E x p e r i m e n t a l A n i m a l s For mammals to be used for studies of cerebral glucose/energy metabolism, maintenance of steady-state conditions (see previous) is a necessary condition. This holds for monkeys, dogs, cats, rats, gerbils, and mice, which have been shown to be appropriate for such studies. Substrate utilization rates, compound concentrations, and enzyme activities can be measured.
Utilization o f Substrates In principle, the same procedures are valid as have been noted previously for human beings. To study global cerebral metabolic rates of oxygen and glucose, the modified Kety-Schmidt technique (9, 10) can be applied in monkeys, dogs, and cats in the same way as in humans (see above), except that mixed cerebral venous blood has to be sampled from the exposed superior sagittal sinus in its distal part close to the confluence of the sinuses. In smaller animals (rats, mice, and gerbils, in particular), the extraction rates when blood is drawn either from an artery or from the superior sagittal sinus are 0.25 ml/min (rat) and 0.05 ml/min (mouse, gerbil) to avoid hemorrhagic shock situations. Local cerebral glucose consumption can be determined by autoradiography with [2-14C]deoxyglucose (6). The labeled tracer is injected intravenously, and arterial blood samples are collected at short intervals over 45 minutes to measure [2-14C]deoxyglucose and glucose concentrations. After the final arterial blood sample at 45 minutes, the animal is decapitated, and the brain is rapidly removed and frozen in 2-methylbutane chilled to - 4 0 to -50~ The frozen brains are coated with chilled embedding medium and sectioned at 20/xm in a cryostat at -22~ The tissue sections are thawmounted on glass coverslips and dried on a hot plate at 60~ Local tissue concentrations of 14C are determined by densitometric analysis of the autoradiograms with a densitometer equipped with a 0.2-mm aperture.
Tissue Studies in Vivo To investigate the metabolic compounds of the glucose/energy metabolism in cerebral tissue, another prerequisite has to be fulfilled: freezing of the brain in situ during maintenance of steady-state conditions (see previous).
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For this purpose, a scalp skin funnel is formed by a sagittal incision and blunt preparation from the skull. Before freezing, a wet, warmed (37~ sponge is put into the funnel to avoid cooling of the brain. Freezing is performed by pouring liquid nitrogen into the funnel for at least 3 minutes in rat, gerbil, and mouse, and for at least 5 minutes in monkey, dog, and cat. After disconnection of the respiratory tube, the whole animal (rat, mouse, gerbil) or only the head of the animal (monkey, dog, cat) is immersed in liquid nitrogen. This freezing technique and the subsequent preparation steps (see following) yield optimal conditions for blockade of catabolic steps in the metabolism of substrates and enzymes in cerebral glucose/energy metabolism; these procedures are also superior to freezing by immersion of the whole animal or the animal head in liquid nitrogen after disconnection of the ventilation tube, which leads to autolytic changes in metabolite concentrations (20, 21). During the subsequent preparation of the cerebral tissue, permanent cooling is necessary. Therefore, the brain as a whole is chiseled from the skull under liquid nitrogen. If regional determinations are to be performed, the brain can be sliced in a cryostat between - 2 0 and -15~ At temperatures higher than - 15~ the tissue metabolism is jeopardized by autolytic variations. The tissue samples can be stored at -80~ for months until biochemical analysis. This technique of brain tissue freezing and preparation is superior to the freeze-blowing technique (22), which does not allow separation of different brain areas or of gray and white matter. As was mentioned previously, the mammalian brain is a multiorgan organ, which means that different areas of the brain fulfill distinct functions because of a highly specialized metabolism. The freeze-blowing technique freezes brain tissue very rapidly, but it is mixed and contaminated with blood, blood vessels, and meninges.
Biochemical Analyses To determine the concentrations of the substrates of the glycolytic chain, the tricarboxylic acid cycle, and the energy pool, the frozen tissue samples are (rapidly) weighed at 4~ homogenized at -28~ in chloroform by an Ultraturrax, and deproteinated with 20 volumes of HC104/EDTA at -28~ The homogenate is centrifuged at 25,000 g for 10 minutes at 0~ Supernatants are neutralized to pH 7.2 with 0.4 M imidazole base, 1.5 N KOH, and 0.3 M KC1. Adenine nucleotides and creatine phosphate can be determined spectrophotometrically at 340 nm (23, 24) or by means of high-performance liquid chromatography (25). The latter technique allows the additional determination of guanine nucleotides and nicotinamide-adenine dinucleotide at 210 nm, but leaves the corresponding monophosphates undetermined. In the
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homogenation procedure of this technique, EDTA imidazole base and KC1 are not added, because these compounds are detected at 210 nm. The metabolites of the glycolytic chain and the tricarboxylic acid cycle are determined spectrophotometrically as described by Bergmeyer (23).
NMR
Spectrometry
As in studies in humans (see previous), energy-rich phosphates can be mapped in brain tissue of experimental animals using surface coils (26). In contrast to studies in humans, the coil can be directly positioned on the surface of the cerebral cortex, so that the spectra are not contaminated by spectra from the extracerebral tissues (skin, bone). This technique has the advantage that from one cerebral cortical area of an anesthetized animal with its head held in a stereotaxic apparatus, numerous data can be collected under different experimental conditions during the course of a single experiment. When studies in different areas are to be performed, the same experimental conditions of tissue freezing (see previous) must be observed. 3~p NMR signals can be detected by the ex vivo in vitro technique: briefly, deeply frozen cerebral tissue is chiseled from the skull under liquid nitrogen, and chipped into pieces of an appropriate size (2.5 g). The NMR signals can be detected at -10~ (27). When labeled [1-~3C]glucose is given by infusion, the glucose metabolism can be investigated in the same manner as can the 31p metabolism (28).
Enzyme Activities
The tissue is prepared in the same way as for substrate determination, i.e., steady-state conditions are maintained and the brain is frozen in situ. The activity rates of the enzymes measured ex vivo in vitro can therefore be assumed to represent the in vivo situation. Because liquid nitrogen abolishes the metabolic activity completely within a few seconds, the activity state of the enzymes may reflect the activity state at the time of tissue freezing. The simultaneous determination of the activities of enzymes and the concentrations of substrates makes it possible to evaluate what proportion of the substrate is complexed by the respective enzyme. For the measurement of enzymatic activities, the frozen tissue samples are homogenized in a 0.02 M Tris-HCl buffer (1 : 10, w/v) containing 0.1 mM EDTA, 0.1 mM DTT, 250 mM sucrose, 100 txl of 10% Triton X-100 at pH 7.5 and 0~ The homogenate is centrifuged at 100,000 rpm for 15 minutes at 2~ The enzymatic activity is determined by continuous optical tests at
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340 nm and 30~ by use of microcuvettes with a final volume of 1 ml. (For the biochemical tests used for the individual determination of the enzyme activities, the reader is referred to Ref. 23.)
Miscellaneous Aspects Anesthesia The maintenance of steady-state conditions (see previous) and the avoidance of painful stimuli, operation stress, and autonomic reflexes require adequate anesthesia in studies of experimental animals. Intraperitoneal administration of anesthetic drugs may influence the cerebral glucose/energy metabolism in an undesirable way, because the stage of anesthesia can hardly be controlled. Intravenous anesthesia with barbiturates, neuroleptics, or ketamine can be better regulated, allowing better maintenance of the steady-state conditions. However, these compounds have been found to cause variations in both CBF and the cerebral oxygen utilization rate in different ways, which means that undesirable and incalculable effects can occur in cerebral glucose/energy metabolism. Inhalation anesthesia with 0.5% (or less) halothane and nitrous oxide/ oxygen (70 : 30, v/v) may be assumed to be appropriate in animal experiments requiring steady-state conditions. The anesthetics are controlled best by a respirator adaptable to frequency and volume of the inspired gas mixture, which is applied via a tracheal tube. In such cases the animal is immobilized by a muscle-relaxing drug because of the restraint. Nitrous oxide in the concentration mentioned above has not been found to influence either cortical CBF or overall glucose utilization of the brain (29, 30). Although halothane in as low a concentration as 0.6% (v/v) reduces the cerebral metabolic rate of oxygen by 25%, no variations in glycolytic flux or in cerebral energy state, except for a decrease in the glucose concentration, could be observed under anesthesia with 1% (v/v) halothane (31). It is even possible to discontinue halothane anesthesia with the onset of steady state without inducing variations in the steady-state parameters.
Aging Process Before the effect of the aging process on cerebral glucose/energy metabolism can be evaluated, the term "age" has to be clearly defined. Rodents may be designated as aged when their strain has a 50% survival rate and when their survival curve is more or less rectangular (32). This definition is also
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valid for other species of experimental animals. It has to be taken into account that the deflection point of any survival curve differs from strain to strain of a species and between the sexes. General guidelines for age-related studies are recommended (33).
Conclusion Various techniques are available for the study of cerebral glucose/energy metabolism in vivo in humans and experimental (mammalian) animals in a reproducible manner. With due consideration for a few limiting conditions, such as steady-state parameters, anesthesia, and the methodologic limitations, important information can be gained on the basis of cellular work in normal and diseased states.
References 1. S. Hoyer, J. Hamer, E. Alberti, H. Stoeckel, and F. Weinhardt, Pfliigers Arch. Ges. Physiol. 351, 161-172 (1974). 2. J. Hamer, S. Hoyer, H. Stoeckel, E. Alberti, and F. Weinhardt, Acta Neurochir. 28, 95-110 (1973). 3. E. Alberti, S. Hoyer, J. Hamer, H. Stoeckel, P. Packschiess, and F. Weinhardt, Br. J. Anaesth. 47, 941-947 (1975). 4. J. Hamer, S. Hoyer, E. Alberti, and F. Weinhardt, Acta Neurochir. 33, 141150 (1976). 5. K. Norberg and B. K. Siesj6, Brain Res. 86, 31-44 (1975). 6. B. K. Siesj6 and L. Nilsson, Scand. J. Clin. Lab. Invest. 27, 83-96 (1971). 7. P. Della Porta, A. T. Maiolo, V. V. Negri, and E. Rossella Metabolism 13, 131-140 (1964). 8. L. D. Lewis, B. Ljunggren, R. A. Ratcheson, and B. K. Siesj6, J. Neurochem. 23, 673-679 (1974). S. S. Kety and C. F. Schmidt, J. Clin. Invest. 27, 476-483 (1948). 10. A. Bernsmeier and K. Siemons, Pfliigers Arch. Ges. Physiol. 258, 149-162 (1953). 11. N. A. Lassen and O. Munck, Acta Physiol. Scand. 33, 30-49 (1955). 12. L. Sokoloff, M. Reivich, C. Kennedy, M. H. DesRosiers, C. S. Patlack, K. D. Pettigrew, O. Sakurada, and M. Shinohara, J. Neurochem. 28, 897-916 (1977). 13. M. Reivich, D. Kuhl, A. Wolf, J. Greenberg, M. Phelps, T. Ido, V. Casella, J. Fowler, E. Hoffman, A. Alavi, P. Som, and L. Sokoloff, Circ. Res. 44, 127-137 (1979). 14. A. Gjedde, Brain Res. Rev. 4, 237-274 (1982). 15. K. Wienhard, G. Pawlik, K. Eriksson, H. W. Ilsen, H. Herholz, and W. D. Heiss, J. Cereb. Blood Flow Metab. 3(1), $474-$475 (1983). .
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PARADIGMSOF NEURAL INJURY 16. R. S. J. Frackowiak, G. L. Lenzi, T. Jones, and J. D. Heather, J. Comput. Assist. Tomogr. 4, 727-736 (1980). 17. A. A. Lammertsma, T. Jones, R. S. J. Frackowiak, and G. L. Lenzi, J. Comput. Assist. Tomogr. 5, 544-550 (1981). 18. D. I. Hoult, S. J. W. Busby, D. G. Gadian, G. K. Radda, R. E. Richards, and P. J. Seeley, Nature (London) 252, 285-287 (1974). 19. J. W. Prichard, J. R. Alger, K. L. Behar, O. A. C. Petroff, and R. G. Shulman, Proc. Natl. Acad. Sci. U.S.A. 80, 2748-2751 (1983). 20. U. Ponten, R. A. Ratcheson, and B. K. Siesj6, J. Neurochem. 21, 1121-1126 (1973). 21. U. Ponten, R. A. Ratcheson, L. G. Salford, and B. K. Siesj6, J. Neurochem. 21, 1127-1138 (1973). 22. R. L. Veech, R. L. Harris, D. Veloso, and E. H. Veech, J. Neurochem. 20, 183-188 (1973). 23. H. U. Bergmeyer, "Methods of Enzymatic Analysis," 2nd Ed. Academic Press, New York, 1974. 24. J. Folbergrova, V. MacMillan, and B. K. Siesj6, J. Neurochem. 19, 24972505 (1972). 25. E. Harmsen, P. P. H. De Tombe, and J. W. De Jong, J. Chromatogr. 230, 131-136 (1982). 26. J. J. H. Ackerman, T. H. Grove, G. G. Wong, D. G. Gadian, G. K. Radda, Nature (London) 283, 167-170 (1980). 27. B. Chance, Y. Nakase, M. Bond, J. S. Leigh, Jr., and G. McDonald, Proc. Natl. Acad. Sci. U.S.A. 75, 4925-4929 (1978). 28. R. G. Shulman, T. R. Brown, K. Ugurbil, S. Ogawa, S. M. Cohen, and J. A. den Hollander, Science 205, 160-166 (1979). 29. N. Dahlgreen, M. Ingvar, H. Yokoyama, and B. K. Siesj6, J. Cereb. Blood Flow Metab. 1, 211-218 (1981). 30. M. Ingvar and B. K. Siesj6, J. Cereb. Blood Flow Metab. 2, 481-486 (1982). 31. L. Nilsson and B. K. Siesj6, J. Neurochem. 23, 29-36 (1974). 32. C. F. Hollander, M. J. van Zwieten, and C. Zurcher, in "Aging of the Brain" (W. H. Gispen and J. Traber, eds.), pp. 187-196. Elsevier, Amsterdam, 1983. 33. P. Coleman, C. Finch, and J. Joseph Neurobiol Aging 11, 1-2 (1990).
Cerebral Glucose/Energy Metabolism" Valid Techniques in Humans and Animals Siegfried Hoyer
Introduction The healthy, mature, nonstarved mammalian brain uses glucose only by oxidizing it to obtain energy in the form of ATP, which is necessary to maintain cellular function. A study of the metabolic pathway of glucose and the energy pool in the brain is of interest in evaluating both normal function and pathological states. The normal supply of the substrates oxygen and glucose to the brain is guaranteed by the blood flow in the carotid and vertebral arteries. Cerebral blood flow (CBF) and cerebral oxidative metabolism have been found to be tightly linked functionally. Therefore, it seems necessary also to touch on the control mechanisms of CBF. The mammalian brain is a multiorgan organ; that is to say, it has a heterogeneous structure and consists of functionally different regions. This holds true for the gray matter in the cerebral cortex and for the diverse subcortical nuclei. Cerebral gray and white matter have to be regarded as distinct from one another in functional terms. It is as yet not known whether the white matter is also characterized by a regional diversity similar, in metabolic terms, to that of the gray matter.
Control Mechanisms of Cerebral Blood Flow There are two physiological parameters that control CBF: (1) the mean arterial blood pressure (MABP) and the cerebral perfusion pressure (CPP) and (2) the partition pressure of carbon dioxide in arterial blood (paCO2). Under normal conditions, MABP is around 100 mmHg in humans and in mammals (monkey, dog, cat, rat) commonly used for experimental purposes. Normal CBF is maintained over an MABP range of around 50 to around 150 mmHg (autoregulation of CBF). However, the cerebral metabolic rates of glucose, lactate, and CO2 change under moderate arterial hypotension (1). The relationship between MABP and CBF is valid only when the intracranial pressure (ICP) is normal (close to zero). When ICP is increased as a result 124
Methods in Neurosciences, Volume 30 Copyright 9 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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of brain edema or other space-occupying lesions, the relationship between CPP and CBF becomes valid (CPP = MABP - ICP) (2). The physiological, normocapnic paCO2 is around 40 mmHg. Hypocapnia induces a fall and hypercapnia induces an increase in CBF in a rather linear manner between paCO 2 of around 20 and 80 mmHg. In this range, the cerebral metabolic rates of oxygen, CO2, glucose, and lactate are kept constant by an increase in the extraction rates of the substrates and vice versa (3).
Physiological Steady State In studies designed to investigate parameters of the cerebral glucose and oxidative energy metabolism, MABP and paCO 2 have to be maintained in physiological ranges. MABP can be monitored either by means ofa sphygmomanometer (preferred method for humans) or via an arterial catheter and pressure transducers (preferred method for experimental animals). Although the arterial partition pressures of oxygen (paO2) do not control CBF, a fall from normoxemic (around 100 mmHg) to hypoxemic values (around 80 mmHg or lower) has been shown to induce changes in cerebral glucose/energy metabolism (4-6). Another parameter that has to be kept normal is the arterial glucose concentration. Arterial hypoglycemia induces a more marked reduction in cerebral glucose consumption than in oxygen utilization (7), and drastic changes in energy metabolism in the cerebral cortex (8). Besides the arterial blood constituents pCO2, PO2, and glucose, further parameters are of importance because of their capability to vary CBF and thus, secondarily, cerebral oxidative metabolism: hematocrit, hemoglobin, and pH. To complete the steady-state parameters, body temperature (37~ has to be recorded either rectally (humans, experimental animals) or intraperitoneally (experimental animals). It is generally accepted that the maintenance of the above steady-state condition over an experimental period of at least 15 minutes guarantees the comparability of different studies. However, in human disease states, or in defined pathophysiological experimental conditions, both quality and quantity of the variations in the respective steady-state parameter will have to be strictly maintained.
Studies in Humans Various techniques are available for the investigation of global and local cerebral utilization rates of oxygen and glucose.
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Kety-Schmidt Technique Global measurements of substrate consumption of any organ are based on the Fick principle: the amount of a substrate that is used by that organ is represented by the difference in the concentrations measured in the arterial blood being supplied to it and in the venous blood released from its, i.e., the arteriovenous difference. However, there are conditions that have to be fulfilled for this principle to hold: the arterial and venous concentrations of the substrates and the blood flow through the organ have to be constant during the period of measurement (steady-state condition; see above), and one main vein only should drain the blood from the organ. The consumption or release of a substrate can then be calculated from the arteriovenous substrate difference and blood flow (9). It can be assumed that any substrate concentration is identical in the blood of all larger arterial vessels of the body. Therefore, there is no need to sample arterial blood from the carotid artery; it can be more conveniently sampled from the femoral artery, for example. The main venous drainage from the brain takes place via the internal jugular vein. However, this vein is joined by the facial, lingual, pharyngeal, and thyroid veins, so that the extracerebral contamination is considerable. Proximal to these veins, the superior bulb of the internal jugular vein is situated in the posterior part of the jugular foramen. Mixed cerebral venous blood, i.e., blood from both cerebral hemispheres, the brain stem, and the cerebellum, with only minor extracerebral contamination, can be collected from the superior bulb of the internal jugular vein by direct puncture. A modification (10) of the original Kety-Schmidt technique (9) facilitates blood sampling in that integral concentrations of blood substrates are collected over 10 minutes by motor syringes extracting 1 ml/ minute of blood, to avoid extracerebral contamination at the venous site in particular. With respect to the measurement of global CBF it must be borne in mind that the tracer used to calculate blood flow must be nontoxic, must not produce any side effects, must not be varied metabolically or itself induce variations in metabolism, should be insoluble or only slightly soluble in blood and adipose tissue, should diffuse rapidly into the brain, should be easily analyzed, should have a known brain-blood partition coefficient, and should be economical in use. Some candidates largely fulfill these prerequisites" nitrous oxide, argon, and both labeled krypton and xenon. Nitrous oxide in low concentration (15-20% in air) is most frequently used, although the calculation of CBF from a 10-minute saturation period is overestimated by 10-15%. This error can be minimized by prolonging the saturation period to 14 minutes and extrapolating to infinity (11). Although this Kety-Schmidt technique, which allows global measurements only, has some limitations, it
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has the advantage of providing data on both CBF and the utilization rates of substrates of interest. Several very sensitive analytical techniques are available for the measurement of cerebral arteriovenous substrate differences, such as oxygen and CO2 (gas chromatography), glucose, lactate, pyruvate, and ketone bodies (spectrophotometry), amino acids (high-performance liquid chromatography), free fatty acids (gas chromatography), etc. Uptake or release of these substrates and the shifts in the pattern of different metabolic pathways can be followed. Furthermore, the duration of one study is around 30-40 minutes from the puncture of the vessels to removal of the needles. It may be easier to maintain a steady state over this period than throughout longer lasting investigations (see following), which makes this method more practical for use in mentally deranged people. Although this technique is invasive, it can be applied almost daily if necessary without causing excessive stress to the patient. Finally, the costs are quite low.
Positron E m i s s i o n T o m o g r a p h y T e c h n i q u e s Positron emission tomography (PET) techniques are based on a modified Fick principle (see previous) in which radioactive tracers are used to determine the cerebral utilization rates of substrates. The direct measurement of radioactivity in the brain tissue replaces the determination of the substrate concentration in the mixed venous blood of the superior bulb of the internal jugular vein. Techniques are developed to investigate the utilization rates of glucose and oxygen. Tracers used are 18F-labeled a-fluoro-deoxyglucose (FDG), [llC]methyl-D-glucose, and 150, The FDG method is the one most commonly used in studies of regional glucose utilization by means of PET. This method is based on the principles outlined by Sokoloff et al. (12) and Reivich et al. (13). Deoxyglucose uses the same carrier at the blood-brain barrier for transport from arterial blood into the brain tissue. Here it is phosphorylated by hexokinase but not converted further into fructose 6-phosphate. Deoxyglucose 6-phosphate is trapped in the brain, and its resulting accumulation is the marker to be quantified by PET. Deoxyglucose 6-phosphate is not a substrate for glucose6-phosphate dehydrogenase, but can be hydrolyzed by glucose-6-phosphatase. For calculation of the local metabolic rate of glucose, the time interval between injection and PET measurement, the plasma concentrations of glucose and FDG during the study, the rate constants of the transmembranous carrier system, of the FDG phosphorylation step and the "lumped constant" must all be known. For studies under normal conditions, the rate constants and the lumped constant can be derived from animal experiments. However,
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under pathologic conditions, individual determination of the rate constants by dynamic PET scanning is thought to be more accurate (14, 15). The determination of local cerebral oxygen consumption is more crucial because of the very short half-life of oxygen-15, which is 2.1 minutes. Oxygen- 15 is inhaled and is almost entirely metabolized to labeled water (H2150). Three emission scans have to be carried out during the inhalation period for quantitative measurement of the metabolic rate of oxygen (16, 17). The immobility of the subject's head is a crucial factor for any PET study. Because the duration of one investigation either of local glucose utilization or of local oxygen consumption is around 1 hour, maintenance of the steady state may become difficult, especially because the patients concerned are often (mentally) restless and uncooperative (demented). Clearly, the PET technique can yield very precise information on regional oxidative metabolism and thus on pathobiochemical pathways in diseased states. Otherwise, an unequivocal disadvantage lies in the high costs (cyclotron, PET) and in the availability of highly trained specialists. Therefore, the availability of such equipment is restricted to a few research centers.
Nuclear Magnetic Resonance In recent years, high-resolution nuclear magnetic resonance (NMR) spectroscopy has been established as a noninvasive technique to investigate phosphorus resonances from brain tissue (18, 19). However, technical difficulties and instrumental limitations still restrict the wide application of NMR spectroscopy. Experimental data so far available on normal and diseased states of cerebral metabolism are characterized by considerable scatter.
Postmortem Studies Postmortem studies of cerebral glucose/energy metabolism are limited to compounds that do not vary at all, or hardly at all, during ischemia/anoxia of the brain in the agonal state. This precludes the reproducible investigation of labile phosphates. Apart from these, enzyme activities of the glycolytic and oxidative glucose breakdown processes (see following) can be validly studied during the first 2 days after death. However, interpretation of the results has to be adapted to allow for the postmortem changes occurring as a result of room temperature and the time lapse before tissue sampling after death. To this end, relevant animal experiments have to be performed to test how and to what extent room temperature and temperature in the cool
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chamber, both for different durations, affect the activities of the enzymes to be studied.
S t u d i e s in E x p e r i m e n t a l A n i m a l s For mammals to be used for studies of cerebral glucose/energy metabolism, maintenance of steady-state conditions (see previous) is a necessary condition. This holds for monkeys, dogs, cats, rats, gerbils, and mice, which have been shown to be appropriate for such studies. Substrate utilization rates, compound concentrations, and enzyme activities can be measured.
Utilization o f Substrates In principle, the same procedures are valid as have been noted previously for human beings. To study global cerebral metabolic rates of oxygen and glucose, the modified Kety-Schmidt technique (9, 10) can be applied in monkeys, dogs, and cats in the same way as in humans (see above), except that mixed cerebral venous blood has to be sampled from the exposed superior sagittal sinus in its distal part close to the confluence of the sinuses. In smaller animals (rats, mice, and gerbils, in particular), the extraction rates when blood is drawn either from an artery or from the superior sagittal sinus are 0.25 ml/min (rat) and 0.05 ml/min (mouse, gerbil) to avoid hemorrhagic shock situations. Local cerebral glucose consumption can be determined by autoradiography with [2-14C]deoxyglucose (6). The labeled tracer is injected intravenously, and arterial blood samples are collected at short intervals over 45 minutes to measure [2-14C]deoxyglucose and glucose concentrations. After the final arterial blood sample at 45 minutes, the animal is decapitated, and the brain is rapidly removed and frozen in 2-methylbutane chilled to - 4 0 to -50~ The frozen brains are coated with chilled embedding medium and sectioned at 20/xm in a cryostat at -22~ The tissue sections are thawmounted on glass coverslips and dried on a hot plate at 60~ Local tissue concentrations of 14C are determined by densitometric analysis of the autoradiograms with a densitometer equipped with a 0.2-mm aperture.
Tissue Studies in Vivo To investigate the metabolic compounds of the glucose/energy metabolism in cerebral tissue, another prerequisite has to be fulfilled: freezing of the brain in situ during maintenance of steady-state conditions (see previous).
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For this purpose, a scalp skin funnel is formed by a sagittal incision and blunt preparation from the skull. Before freezing, a wet, warmed (37~ sponge is put into the funnel to avoid cooling of the brain. Freezing is performed by pouring liquid nitrogen into the funnel for at least 3 minutes in rat, gerbil, and mouse, and for at least 5 minutes in monkey, dog, and cat. After disconnection of the respiratory tube, the whole animal (rat, mouse, gerbil) or only the head of the animal (monkey, dog, cat) is immersed in liquid nitrogen. This freezing technique and the subsequent preparation steps (see following) yield optimal conditions for blockade of catabolic steps in the metabolism of substrates and enzymes in cerebral glucose/energy metabolism; these procedures are also superior to freezing by immersion of the whole animal or the animal head in liquid nitrogen after disconnection of the ventilation tube, which leads to autolytic changes in metabolite concentrations (20, 21). During the subsequent preparation of the cerebral tissue, permanent cooling is necessary. Therefore, the brain as a whole is chiseled from the skull under liquid nitrogen. If regional determinations are to be performed, the brain can be sliced in a cryostat between - 2 0 and -15~ At temperatures higher than - 15~ the tissue metabolism is jeopardized by autolytic variations. The tissue samples can be stored at -80~ for months until biochemical analysis. This technique of brain tissue freezing and preparation is superior to the freeze-blowing technique (22), which does not allow separation of different brain areas or of gray and white matter. As was mentioned previously, the mammalian brain is a multiorgan organ, which means that different areas of the brain fulfill distinct functions because of a highly specialized metabolism. The freeze-blowing technique freezes brain tissue very rapidly, but it is mixed and contaminated with blood, blood vessels, and meninges.
Biochemical Analyses To determine the concentrations of the substrates of the glycolytic chain, the tricarboxylic acid cycle, and the energy pool, the frozen tissue samples are (rapidly) weighed at 4~ homogenized at -28~ in chloroform by an Ultraturrax, and deproteinated with 20 volumes of HC104/EDTA at -28~ The homogenate is centrifuged at 25,000 g for 10 minutes at 0~ Supernatants are neutralized to pH 7.2 with 0.4 M imidazole base, 1.5 N KOH, and 0.3 M KC1. Adenine nucleotides and creatine phosphate can be determined spectrophotometrically at 340 nm (23, 24) or by means of high-performance liquid chromatography (25). The latter technique allows the additional determination of guanine nucleotides and nicotinamide-adenine dinucleotide at 210 nm, but leaves the corresponding monophosphates undetermined. In the
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homogenation procedure of this technique, EDTA imidazole base and KC1 are not added, because these compounds are detected at 210 nm. The metabolites of the glycolytic chain and the tricarboxylic acid cycle are determined spectrophotometrically as described by Bergmeyer (23).
NMR
Spectrometry
As in studies in humans (see previous), energy-rich phosphates can be mapped in brain tissue of experimental animals using surface coils (26). In contrast to studies in humans, the coil can be directly positioned on the surface of the cerebral cortex, so that the spectra are not contaminated by spectra from the extracerebral tissues (skin, bone). This technique has the advantage that from one cerebral cortical area of an anesthetized animal with its head held in a stereotaxic apparatus, numerous data can be collected under different experimental conditions during the course of a single experiment. When studies in different areas are to be performed, the same experimental conditions of tissue freezing (see previous) must be observed. 3~p NMR signals can be detected by the ex vivo in vitro technique: briefly, deeply frozen cerebral tissue is chiseled from the skull under liquid nitrogen, and chipped into pieces of an appropriate size (2.5 g). The NMR signals can be detected at -10~ (27). When labeled [1-~3C]glucose is given by infusion, the glucose metabolism can be investigated in the same manner as can the 31p metabolism (28).
Enzyme Activities
The tissue is prepared in the same way as for substrate determination, i.e., steady-state conditions are maintained and the brain is frozen in situ. The activity rates of the enzymes measured ex vivo in vitro can therefore be assumed to represent the in vivo situation. Because liquid nitrogen abolishes the metabolic activity completely within a few seconds, the activity state of the enzymes may reflect the activity state at the time of tissue freezing. The simultaneous determination of the activities of enzymes and the concentrations of substrates makes it possible to evaluate what proportion of the substrate is complexed by the respective enzyme. For the measurement of enzymatic activities, the frozen tissue samples are homogenized in a 0.02 M Tris-HCl buffer (1 : 10, w/v) containing 0.1 mM EDTA, 0.1 mM DTT, 250 mM sucrose, 100 txl of 10% Triton X-100 at pH 7.5 and 0~ The homogenate is centrifuged at 100,000 rpm for 15 minutes at 2~ The enzymatic activity is determined by continuous optical tests at
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340 nm and 30~ by use of microcuvettes with a final volume of 1 ml. (For the biochemical tests used for the individual determination of the enzyme activities, the reader is referred to Ref. 23.)
Miscellaneous Aspects Anesthesia The maintenance of steady-state conditions (see previous) and the avoidance of painful stimuli, operation stress, and autonomic reflexes require adequate anesthesia in studies of experimental animals. Intraperitoneal administration of anesthetic drugs may influence the cerebral glucose/energy metabolism in an undesirable way, because the stage of anesthesia can hardly be controlled. Intravenous anesthesia with barbiturates, neuroleptics, or ketamine can be better regulated, allowing better maintenance of the steady-state conditions. However, these compounds have been found to cause variations in both CBF and the cerebral oxygen utilization rate in different ways, which means that undesirable and incalculable effects can occur in cerebral glucose/energy metabolism. Inhalation anesthesia with 0.5% (or less) halothane and nitrous oxide/ oxygen (70 : 30, v/v) may be assumed to be appropriate in animal experiments requiring steady-state conditions. The anesthetics are controlled best by a respirator adaptable to frequency and volume of the inspired gas mixture, which is applied via a tracheal tube. In such cases the animal is immobilized by a muscle-relaxing drug because of the restraint. Nitrous oxide in the concentration mentioned above has not been found to influence either cortical CBF or overall glucose utilization of the brain (29, 30). Although halothane in as low a concentration as 0.6% (v/v) reduces the cerebral metabolic rate of oxygen by 25%, no variations in glycolytic flux or in cerebral energy state, except for a decrease in the glucose concentration, could be observed under anesthesia with 1% (v/v) halothane (31). It is even possible to discontinue halothane anesthesia with the onset of steady state without inducing variations in the steady-state parameters.
Aging Process Before the effect of the aging process on cerebral glucose/energy metabolism can be evaluated, the term "age" has to be clearly defined. Rodents may be designated as aged when their strain has a 50% survival rate and when their survival curve is more or less rectangular (32). This definition is also
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valid for other species of experimental animals. It has to be taken into account that the deflection point of any survival curve differs from strain to strain of a species and between the sexes. General guidelines for age-related studies are recommended (33).
Conclusion Various techniques are available for the study of cerebral glucose/energy metabolism in vivo in humans and experimental (mammalian) animals in a reproducible manner. With due consideration for a few limiting conditions, such as steady-state parameters, anesthesia, and the methodologic limitations, important information can be gained on the basis of cellular work in normal and diseased states.
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