Imaging and the ontogeny of brain metabolism

5 Imaging and the ontogeny of brain metabolism ASTRID N E H L I G

Human brain maturation is incomplete at birth and, during development, the brain undergoes the sequential anatomical, functional and organizational changes necessary to support the complex adaptative behaviour of a fully mature normal subject. Brain development is associated with changes in local cerebral metabolic rates for glucose (LCMRglcs) (Chugani and Phelps, 1986; Chugani et al, 1987; Chugani, 1992) and in local cerebral blood flow (LCBF) (Kennedy and Sokoloff, 1957; Ogawa et al, 1987; Tzourio et al, 1988; Rubinstein et al, 1989; Chiron et al, 1992). In newborn infants, LCMRglcs are low and reach adult levels by 2 years. Thereafter, they further increase to levels 1.5-2.2 times higher in the 3-8-year-old child than in the adult. From 9 years, LCMRglcs decline to reach adult rates by the end of the second decade (Chugani and Phelps, 1986; Chugani et al, 1987; Chugani, 1992). Rates of LCBF undergo the same postnatal evolution as LCMRglcs with levels 50-85 % higher in the 3-8-year-old child than in the adult, followed by a decline to reach adult values by 15-19 years (Chiron et al, 1992). These data on regional maturation of human brain glucose utilization and blood flow confirm the relationship between the metabolic and circulatory increase within neuroanatomical structures and the emergence of corresponding functions, essentially during the first year of life (Chiron et al, 1992; Chugani and Phelps, 1986; Chugani et al, 1987; Chugani, 1992). The high energy demand and blood flow between 3 and 8 years is essentially related to the excessive fuel expenditure related to normal brain cellular growth and myelination (Chugani et al, 1987). Glucose is the major metabolic substrate, almost exclusively the sole one, for the human brain at all ages. This assumption is supported by the measurement of cerebral arteriovenous differences for both glucose and oxygen in term and preterm infants (Kraus et al, 1974). However, in spite of the close relationship between LCMRglcs and the emergence of specific cerebral functions, there is still no clear consensus to define whether early hypoglycaemia may be significantly correlated with compromised neurodevelopmental outcome (Cornblath et al, 1990). Therefore, the present review will be focused on the postnatal evolution of substrate use by the rat brain determined by autoradiographic techniques. Indeed, although blood glucose levels in the infant rat are close to those of adults, the infant rat brain is in a kind of hypoglycaemic situation with a limited supply of glucose to the brain, because of the relative inefficiency of the glucose transporter at early Bailli~re' s Clinical Endocrinology and Metabolism--

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ages and because of the lack of development of oxidative glucose metabolism enzymes. The relatively low level of glucose utilization by the immature rat brain is compensated by a transient active utilization of ketone bodies during the suckling period (for review, see Nehlig and Pereira de Vasconcelos, 1993).

GLUCOSE AND OXYGEN CONSUMPTION OF THE IMMATURE RAT BRAIN AS A WHOLE Both oxygen and glucose consumption of the whole rat brain undergo a sigmoid rise between birth and adulthood. Indeed, the oxygen uptake of the brain is lowest during the first week of life, reaches its greatest level between the 4th and the 7th week and then falls off gradually by 10-15% to reach the adult level at about 20 weeks (Himwich et al, 1939; Tyler and Van Harreveld, 1942; Fazekas et al, 1951). Likewise, the postnatal evolution of whole-brain glucose utilization, measured by various techniques, shows the same pattern of changes during postnatal development. Glucose utilization in the rat brain at birth is very low, 2-4 txmol/100 g per min, i.e. about 3-5% of the adult level. Thereafter rates of glucose utilization in the whole brain slowly increase, reaching 17-24 at 10 days and 40-53 ~xmol/100g per min at weaning, 20-21 days. Then, whole-brain glucose utilization levels attain 64 and 67 p.mol/100 g per min in the 35-day-old and adult rat brain, respectively (for review, see Nehlig and Pereira de Vasconcelos, 1993).

REGIONAL UTILIZATION OF GLUCOSE IN THE IMMATURE RAT BRAIN

The measurement of LCMRglcs in the developing rat brain has been assessed by means of the quantitative autoradiographic 2-deoxy[14C]glucose method of Sokoloff et al (1977) applied to the immature rat in our laboratory (Nehlig et al, 1988a,b). The fully quantitative method for estimating local cerebral glucose utilization requires repeated arterial sampling to monitor temporal evolution of plasma glucose and radioactive deoxyglucose levels (Sokoloff et al, 1977). In young rats, frequent blood sampling and intravenous administration of the radioactive tracer through indwelling catheters pose technical problems because of the small size and the fragility of the pups. For those reasons, most authors used qualitative or semiquantitative techniques to measure LCMRglcs in immature rats (Di Rocco and Hall, 1981; Miller, 1986; Richards et al, 1989). However, more recently, several groups have been able to set up the fully quantitative 2-deoxy[14C]glucose technique, with the determination of the rate and lumped constants necessary for the final calculation of LCMRglcs (Sokoloff et al, 1977), in both fetal (Munch et al, 1987; Dyve and Gjedde, 1991) and suckling rats (Nehlig et al, 1988a,b; Vannucci et al, 1989; Bilger et al, 1991; B6mont et al, 1992; E1 Hamdi et al, 1992; Pereira de Vasconcelos et al, 1992).

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Correlation between changes in local cerebral glucose utilization and the acquisition of specific functions LCMRglcs are low and quite homogeneous at the early stages of postnatal development in the rat. They reach values ranging from 9 to 13 txmol/100 g per min at 7 days (Vannucci et al; 1989) and from 30 to 44 Fxmol/100g per min at 17 days after birth in most structures studied (Nehlig et al, 1988a,b). At these early stages, the characteristic features of cerebral glucose utilization are low levels and homogeneity, except in some posterior areas, such as in cerebellar structures at 7 days (Vannucci et al, 1989) and regions of the brainstem from 10 to 17 days (Nehlig et al, 1988a,b). Most of the significant increases in LCMRglc occurring between 10 and 17 days in the rat are of special interest. Indeed, from 10 to 14 days after birth, LCMRglcs significantly increase only in 6 areas out of the 68 studied. Among those are four thalamic and brainstem auditory relay nuclei (Nehlig et al, 1988a,b). Increases in LCMRglc in these auditory areas occur precisely at a period corresponding to the maturation of auditory function and parallel the opening of the auditory meatus normally at 12-13 days, accompanied by a marked increase in the sensitivity to a wide range of tones (Crowley and Hepp-Reymond, 1966; Rose and Ellingson, 1970). Simultaneously, the electrical responses to auditory stimuli appear at about 12-14 days of age (Mourek et al, 1967; Myslivecek, 1970). Moreover, between 14 and 17 days after birth, LCMRglc significantly increases in the auditory cortex, reflecting the ongoing maturation of auditory function at this highest level of integration (Nehlig et al, 1988a,b). In the 15-16-day-old rat, the eyelids usually open and the electroretinogram shows a characteristic pattern (Dowling and Sidman, 1962; Rose and Ellingson, 1970). Visual evoked potentials can be elicited at 14 days, and after 18 days photically evoked discharges are apparent (Klingberg and Schwartze, 1966; Mourek et al, 1967; Myslivecek, 1970). The acquisition of visual function translates into an increase in LCMRglc in the thalamic visual relay nucleus, lateral geniculate body between 14 and 17 days (Nehlig et al, 1988a,b). Furthermore, by 17 days after birth, significant developments in the rat's somatic behaviour are apparent. The capacity of the animals to explore their environment, as well as the variety of their apparently playful activities, is indicative of a more highly organized behaviour that implies the participation of well-differentiated neural connections in the neocortex, especially in the parietal area (Tilney, 1933). These behavioural changes translate into metabolic increases between 14 and 17 days in the frontoparietal cortex, both the somatosensory and motor areas, as well as in limbic and motor thalamic relay nuclei and in posterior motor nuclei (Nehlig et al, 1988a,b). By weaning time (21 days), most of the characteristics of the adult rat behaviour are already apparent. All parts of the neocortex have by this time attained a degree of differentiation sufficient to make them able to receive somesthetic, auditory and visual impulses (Tilney, 1933). This high degree of cerebral development translates into a general increase in LCMRglc ranging from 50 to 100% in all cerebral structures. By 21 days, the average

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rate of cerebral glucose utilization reaches 76% of the adult value, and LCMRglcs start to differentiate and to reach some level of heterogeneity within the brain (Figure 1) (Nehlig et al, 1988a,b). These data are in good agreement with studies on the development of LCMRglc in human brain, especially during the first year of life (Chugani and Phelps, 1986; Chugani et al, 1987; Chugani, 1992). Indeed, in infants 5 weeks of age, LCMRglcs are highest in the sensorimotor cortex, thalamus, midbrain-brainstem and cerebellar vermis, underlining the predominantly subcortical and primitive sensorimotor level of function at that age. By 3 months, LCMRglcs increase in the parietal, temporal and occipital cortices, and in the basal ganglia. At that time, many of the intrinsic subcortical reflexes are being suppressed and more coordinated motions as well as visuosensorimotor integration are appearing. By 8 months, subsequent increases in LCMRglc occur in frontal cortex and various associative regions and are accompanied by the appearance of higher cortical and cognitive function, and by more meaningful interaction with surroundings (Chugani and Phelps, 1986; Chugani et al, 1987; Chugani, 1992). Thus, it appears that increases in LCMRglcs in the human and rat infant are in good agreement with behavioural, neurophysiological and anatomical changes known to occur during brain development, as previously shown in other species as well (Kennedy et al, 1972; Abrams et al, 1984). Correlation between local cerebral glucose utilization, electrical and biochemical maturation in the rat brain

The period extending from birth to postnatal day 24 is considered to be a critical period in rat brain development, corresponding to the time of fastest growth, the so-called 'brain growth spurt' period (Dobbing, 1968; Alling, 1985). The advanced state of maturation of the rat brain at weaning time, i.e. 21 days, is reflected by high levels of LCMRglc in all cerebral structures (Nehlig et al, 1988a,b). This age corresponds to the end of the active brain growth spurt, to the peak of myelination and to the adult content of DNA and gangliosides in the rat brain, which indicates the end of cellular growth and synaptogenesis (for review, see Nehlig and Pereira de Vasconcelos, 1993). The marked increase in glucose utilization occurring between 10 and 21 days after birth is accompanied by a parallel rise in the activity of enzymes of the oxidative degradation of glucose in the brain (Sokoloff, 1973; Booth et al, 1980; Leong and Clark, 1984a,b; Nehlig and Pereira de Vasconcelos, 1993) and by a large increase in the rate of conversion of glucose carbon into cerebral amino acids (Gaitonde and Richter, 1966; Cocks et al, 1970). During the same period, brain electrical activity develops. The electroencephalogram first appears at postnatal day 6 and is low and slow in amplitude. It speeds up and increases in voltage near the end of the second postnatal week and takes on the features of adult spectral composition by the end of the third postnatal week (Tuge et al, 1960; Deza and Eidelberg, 1967), when LCMRglcs have already reached quite high levels throughout the brain (Nehtig et al, 1988a,b).

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Evolution of local cerebral glucose utilization between weaning and adulthood

At weaning time, even if some brain metabolic processes and biochemical constituents have reached a level close to the adult level, the rat brain is still undergoing quite active maturation. For example, the cerebral cortex has not attained its full structural specialization by postnatal day 21. The end of the most rapid phase of myelination takes place around 30 days (Norton and Poduslo, 1973). Furthermore, the behaviour of the rat is still changing; playful activities are complete by 35 days, and sexual maturity is attained by 50-60 days (Tilney, 1933). Adult patterns of cortical electrical activity are only reached by 8 weeks (Yoshii and Tsukiyama, 1971; Deza and Eidelberg, 1967). All these maturational changes translate into an increase of 25% in average cerebral glucose utilization between 21 and 35 days, which affects all cerebral functional systems. Conversely, between 35 days and the adult stage, when cerebral growth has plateaued, increases in LCMRglcs are only recorded in scattered areas of the cerebral cortex and in the late-developing hippocampus (Nehlig et al, 1988a,b). Summary

The particularly interesting feature in postnatal development of LCMRglcs in the rat is the transition from very low and almost completely homogeneous levels at postnatal day 10 to the heterogeneous distribution characteristic of the adult rat brain (Figure 1). The heterogeneity in the distribution of cerebral glucose utilization levels already appears at postnatal day 20-21 (Miller, 1986; Nehlig et al, 1988a,b; Richards et al, 1989) and develops further until adulthood (Nehlig et al, 1988a,b). In the adult rat, the ratio between the highest (inferior colliculus, auditory cortex) and the lowest level of glucose utilization (amygdala, hypothalamus) in cerebral grey matter reaches a value of about 4 (Nehlig et al, 1988a; Sokoloff et al, 1977). CEREBRAL KETONE BODY UTILIZATION

Because of the high lipid and low carbohydrate content of maternal milk, the rat pup soon after birth develops a nutritional ketosis that lasts throughout the whole suckling period (for review, see Nehlig and Pereira de Vasconcelos, 1992). During that period, ketone bodies constitute an important proportion (22-76%) of the total energy metabolism balance of the brain and positive cerebral arteriovenous differences are recorded for ~3-hydroxybutyrate and acetoacetate proportional to their concentration in arterial blood (Hawkins et al, 1971; Krebs et al, 1971; Kraus et al, 1974; Schroeder et al, 1991). With compartmental analysis of isotopic data and with measurement of cerebral arteriovenous differences and CBF, the rate of ketone body utilization by the brain of the 18-day-old rat has been found to be equal to a mean of about 30 p.mol/100 g per min (Cremer and Heath, 1974, 1975; Dahlquist and Persson, 1976). These data confirm that ketone

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bodies can account for at least 30% of the total energy metabolism balance in suckling rats (Cremer, 1982). More recently, autoradiographic techniques for the measurement of regional rates of ~-hydroxybutyrate utilization have also been developed, both in the adult (Hawkins and Biebuyck, 1979, 1980) and in the suckling rat brain (Nehlig et al, 1991).

Regional utilization of ketone bodies by the immature rat brain

The regional levels of ketone body uptake and utilization by the immature rat brain have been measured after injection of labelled [3-hydroxybutyrate both in hand-dissected cerebral areas (Cremer, 1980; Miller, 1986) and in autoradiographic brain sections (Nehlig et al, 1991). However, unlike glucose and deoxyglucose, there is no analogue of [3-hydroxybutyrate available whose metabolism in brain tissue would stop after a limited number of enzymatic steps. For these reasons, it is impossible to set up a model for quantitative autoradiographic measurement of cerebral regional [3-hydroxybutyrate utilization. Therefore, semiquantitative analyses are performed, based on the measurement of the amount of radioactivity accumulated in brain regions during a short time, usually 5 rain after labelled ~-hydroxybutyrate injection in order to avoid any significant loss of label from brain tissue. Autoradiographic studies show that utilization of ~3-hydroxybutyrate by the immature rat brain is quite high during the whole suckling period, from 10 to 17 days after birth (Figure 2), reaching peak levels at 14 days (Nehlig et al, 1991). These results are consistent with previous studies reporting that the rate of 13-hydroxybutyrate utilization is highest in brain from 11 to 15 days (DeVivo et al, 1975; Dombrowski et al, 1989). Between 17 and 21 days, regional ~-hydroxybutyrate levels decrease by 50-60% in all cerebral regions (Nehlig et al, 1991). This decrease is concomitant with the marked increase in local cerebral glucose utilization occurring at the same period (Nehlig et al, 1988a,b). Thereafter, from 21 to 35 days, ~-hydroxybutyrate tissular accumulation decreases further, by about 50% in most brain areas (Figure 2), except in cerebral cortex where the decrease is less marked-only 10-20% (Nehlig et al, 1991). These data are consistent with the high permeability of the blood-brain barrier to ketone bodies and the high activity of the enzymes of ketone body metabolism in the suckling rat brain (for review, see Nehlig and Pereira de Vasconcelos, 1993). Conversely to the marked heterogeneity in regional levels of cerebral glucose utilization, there is no evidence of interregional differences in the levels of uptake of the ketone bodies. Ratios between the most highly labelled (vestibular nucleus or inferior colliculus) and the less marked structure (globus pallidus or hypothalamus) reach a value of 1.3-2.0 during suckling (Cremer, 1980; Miller, 1986; Nehlig et al, 1991). This relative interregional homogeneity of ketone body use during suckling is consistent with the important role of ketone bodies as precursors for amino acid and lipid biosynthesis, and thus for membrane and myelin edification (for review, see Nehlig and Pereira de Vasconcelos, 1993). It is then quite likely

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that ketone bodies participate in these biosynthetic processes at a comparable efficiency in all brain regions during early development. At 35 days, the highest levels of labelling are found in cerebral cortex, and the ratio between cortex and hypothalamus is 2.7 (Nehlig et al, 1991). These data agree with the results of Hawkins and Biebuyck (1979, 1980), who noticed in the adult rat a regional heterogeneity in cerebral [~-hydroxybutyrate uptake,-with a higher grain density in the deeper layer of cerebral cortex, which we also recorded at 35 days (Nehlig et al, 1991). The high levels of [3-hydroxybutyrate accumulation in specific regions of the adult rat brain are attributed to a regional heterogeneity in the permeability to ketone bodies (Hawkins and Biebuyck, 1979, 1980), which does not seem to exist in the suckling rat brain when the rates of ketone body transport are very efficient (Cremer et al, 1976, 1979; Moore et al, 1976; Daniel et al, 1977). Summary

In contrast to glucose utilization by the immature rat brain cerebral utilization of ketone bodies is very active during the suckling period when these substrates are supplied in high quantities by circulating blood. However, [3-hydroxybutyrate utilization seems to be rather homogeneous throughout the immature rat brain, which is consistent with active incorporation into amino acids and lipids necessary for membrane and myelin edification. Therefore, in the rat brain, changes in LCMRglc are region-specific and underlie functional changes, as in the human brain, whereas ketone bodies appear to be rather oriented towards cellular edification. Conversely, in the human brain, the active brain growth occurs mainly between 3 and 9 years of age when glucose is the sole cerebral substrate.

CORRELATION BETWEEN ENERGY METABOLISM LEVELS AND BLOOD FLOW IN THE DEVELOPING BRAIN

In the adult brain, levels of LCBF are usually tightly coupled to metabolic demands, so that changes in rates of LCMRglc are accompanied by parallel changes in LCBF rates (DesRosiers et al, 1974; Kuschinsky, 1987; Reivich, 1974; Sokoloff, 1981). In the human infant, the highest levels of both LCMRglc and LCBF occur at the same period, i.e. 3-8 years of life (Kennedy and Sokoloff, 1957; Chugani and Phelps, 1986; Chugani et al, 1987; Rubinstein et al, 1989; Chiron et al, 1992). However, in the rat, the most active brain growth phase takes place before weaning, when the rat is depending on both glucose and ketone bodies as substrates for metabolic and biosynthetic pathways. When average brain levels of glucose and [3-hydroxybutyrate utilization are summed the peak level of energy metabolism appears at 14 days (Nehlig and Pereira de Vasconcelos, 1993), whereas the peak of LCBF is recorded at 17 days (Nehlig et al, 1989a,b). However, in the rat, cerebral energy metabolism and LCBF levels remain coupled during development, with only a change in the level of coupling

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characterized by a relative hypoperfusion ~t 14 days and a relative hyperperfusion at 17 days compared to metabolism (Nehlig and Pereira de Vasconcelos, 1993). It may well be that in the immature brain, given the high metabolic demands necessary for energetic and mainly biosynthetic needs, the adjustments of LCBF to energy needs could be delayed and that the level of coupling between flow and metabolism may vary as a function of age, partly reflecting the immaturity of the brain. Indeed, the delay in the appearance of peak LCBF values compared to energy metabolism rates in the suckling rat is in good accordance with the data from Kennedy et al (1972) in the immature dog. In that species, increases in LCBF correlate, but always with some delay, with the development of sensory and behavioural functions (Kennedy et al, 1972), whereas increases in LCMRglcs correlate in time with the appearance of specific functions (Abrams et al, 1984; Chugani et al, 1987, 1991; Nehlig et al, 1988a,b). However, in humans, while LCMRglcs and LCBF rates are both highest between 3 and 8 years, no precise data are available on the level of coupling between cerebral blood flow and energy metabolism, especially during the first year of life when most behavioural functions are developing. HYPOGLYCAEMIA AND IMAGING OF BRAIN METABOLISM AND BLOOD FLOW

The effects of hypoglycaemia on LCMRglcs have not been studied extensively by the 2-deoxy[14C]glucose method, since the regular operational equation of the method only applies to normoglycaemia. Indeed, the lumped constant that corrects for the slight conformational difference between glucose and deoxyglucose dramatically changes with the level of hypoglycaemia and ranges from 1.2 at an arterial glucose level of 1.9 mmol/1 to 0.48 in normoglycaemia, i.e. at 4.5-6.0 mmol/l of arterial glucose in the rat (Suda et al, 1990). Effects of hypoglycaemia in adult animals

Only minor alterations in LCMRglc occur with plasma glucose concentrations of 2 mmol/1, whereas LCMRglcs decrease in numerous structures of adult rats with severe hypoglycaemia (1.4 mmol/1). The changes are especially significant in structures with normal high metabolic rates, suggesting that glucose delivery and transport to the tissue are rate-limiting (AbdulRahman and Siesj6, 1980; Bryan et al, 1986; Suda et al, 1990). Simultaneously hypoglycaemia is, as are hypoxia and ischaemia, accompanied by cerebral vasodilation and induces widespread increases in LCBF in adult rats, which could reach as much as 400-500% in some regions (Abdul-Rahman et al, 1980; Bryan et al, 1987). During the recovery period, i.e. after glucose injection, hypoperfusion occurs in many brain areas and regions with highly reduced LCMRglc develop a mismatch between blood flow and metabolic rate, with a disproportionate reduction of flow, and

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therefore of oxygen supply, that may contribute to cell damage and neurological deficits (Abdul-Rahman et al, 1980; Abdul-Rahman and Siesj6, 1980). Effects of hypoglycaemia in newborn animals

Autoradiographic techniques have recently been applied to the measurement of LCMRglc and LCBF during hypoglycaemia in newborn dogs. Hypoglycaemia, with blood glucose levels as low as" 0.9 mmol/l, increases LCBF by 170-250% in all regions, whereas, unlike in adults, LCMRglcs are maintained in many structures and decreased only in three regions (Mujsce et al, 1989). The ability of the immature brain to maintain normal LCMRglcs in hypoglycaemia has been partly related to the low cerebral energy requirements relative to adult brain, allowing an increased metabolic and functional tolerance to hypoglycaemia (Mujsce et al, 1989). Moreover, in newborn animals, severe hypoglycaemia is associated with an increase in cerebral lactate utilization. Indeed, lactate is an important metabolic substrate for the brain during the early postnatal period in many species, including humans (Levitsky et at, 1977; Vannucci et al, 1980; Fernandes et al, 1984; Dombrowski et al, 1989; Medina et al, 1990; Young et al, 1991) and can supplement glucose as the primary energy fuel in hypoglycaemia (up to 56% of the total energy metabolism balance), thus preserving a normal CMRo2 (Nemoto et al, 1974; Levitsky et al, 1977; Gardiner, 1980; Hernandez et al, 1980; Vannucci et al, 1981; Hellman et al, 1982; Thurston et al, 1983). Cerebral glucose utilization falls from 93.5% in normoglycaemia to 41% in hypoglycaemia, whereas cerebral lactate use rises from 4% to 58% (Hernandez et al, 1980; Vannucci et al, 1980). Other alternative cerebral fuels, such as ketone bodies actively used by the brain of the immature rat can substitute for glucose during hypoglycaemia in human infants, children and adults (Owen et al, 1967; Persson et al, 1972), but their supply is limited during the newborn period (Settergren et al, 1976; Stanley et al, 1979). This is not the case for lactate, which is elevated above physiological concentrations in the first few days of life (1-2 mmol/1) (Stemberg and Hodr, 1966; Stanley et al, 1979). CONCLUSION These studies show that the newborn brain of various animal species seems relatively protected from the deleterious effects of hypoglycaemia because of both the low energy demand and the possibility of using alternative substrates. Therefore, it is likely that the brain of the human neonate is also, at least partly, protected for the same reasons from deleterious effects of hypoglycaemia on neurological function. However, the longer the hypoglycaemia, especially for several days, the higher the vulnerability (Cornblath et al, 1990). Moreover, data are still lacking on the regional utilization of lactate by the immature brain of both normo- and hypoglycaemic animals. Likewise, it

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would be helpful to study regional utilization of ketone bodies in neonates of an animal species whose brain is, as in humans, already mostly dependent on glucose and not in a state of nutritional ketosis, as in the rat. In fact, we need to define an animal model in which cerebral metabolic activity is closer to that of the human infant brain. This animal model could be the kitten, which has been proposed and used recently to test the biological significance of the LCMRglc maturational curve (Chugani et al, 1991; Chugani, 1992). The effects of hypoglycaemia on glucose utilization in the various brain areas should be studied in that model, in order to define a possible hierarchy in the vulnerability of brain regions to hypoglycaemia. It would also be helpful to control the extent to which lactate and ketone bodies were able to supplement brain energy metabolism, especially in areas with high metabolic demand.

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