Chapter 12. Hypoglycemia and Brain Injury

592



UNIT V



METABOLIC ENCEPHALOPATHIES



220

4.1



200

180



2.5



Plasma glucose (mg/dL)



140

120

100



(52)

*

(35)



80



(51)

(51)



(55)

(69)



(55)



(40)



(49)



60



(26)



Blood glucose concentration (mmol/L)



2.5

160



2.2

1.5

1.6

1.6



1.9

2.6

2.7



(52)

40



6.5

( ) Number of samples

* Mean & 95% confidence interval



20



68



6



-9



2



-7



8



-4



4



-2



-1



97



6



73



3 4



49



2



25



1



12



0



4.1



Age (hr)

Figure 12-1 Neonatal glucose values. Plasma glucose values during

the first week of life in healthy term newborns appropriate for gestational age. (From Srinivasan G, Pildes RS, Cattamanchi G, Voora S,

et al: Clinical and laboratory observations: Plasma glucose values in

normal neonates—a new look, J Pediatr 109:114–117, 1986.)



visual evoked potentials were observed.19 The duration

of hypoglycemia was not determined but was considered

‘‘short.’’19 However, careful epidemiological studies

suggested a deleterious effect on subsequent cognitive

development in infants whose plasma glucose levels

were less than approximately 47 mg/dL on at least one

occasion on 3 or more separate days (Fig. 12-3).20

Abnormalities in arithmetic and motor scores persisted

at 7.5 to 8 years.21 In both the neurophysiological and

the epidemiological studies that suggest a deleterious

effect of hypoglycemia, neonatal neurological signs

were minimal or absent. Thus, clearly duration and

degree of depression of blood glucose are crucial. As discussed later, studies of human infants showed an

increase in cerebral blood flow (CBF) at glucose values

lower than 30 mg/dL and suggested that at a glucose

level lower than approximately 54 mg/dL, transport

becomes limiting for cerebral glucose use. Additional

factors of importance in determination of a critical

threshold value of blood glucose are the rates of CBF

and of cerebral utilization of glucose, as well as the presence

of any alternative substrates. Thus, the threshold

level of blood glucose is different in the infant with

impaired CBF, as with hypotension, or with increased

cerebral glucose utilization, as with seizures or with the



Figure 12-2 Serial brain stem auditory evoked potentials recorded

in a 2-day-old infant in relation to his blood glucose concentration.

The vertical lines indicate the latency between wave I and wave V in the

initial recording during normoglycemia. Note the prolongation of latency

when blood glucose values decreased to 2.5 mmol/L and lower. (From

Koh TH, Aynsley-Green A, Tarbit M, Eyre JA: Neural dysfunction during

hypoglycaemia, Arch Dis Child 63:1353–1358, 1988.)



anaerobic glycolytic metabolism of hypoxia-ischemia

or asphyxia. Currently, I believe that the definition of

hypoglycemia must be individualized according to the

infant’s clinical situation (see ‘‘Management’’ later), and

that the normative statistical data shown in Table 12-1

and Figure 12-1 should be considered only starting

points.

NORMAL METABOLIC ASPECTS

Brain as the Primary Determinant

of Glucose Production

The pathophysiology of neonatal hypoglycemic encephalopathy has as its basis the importance of glucose

as the primary metabolic fuel for brain. Glucose for

normal brain metabolism is derived from the blood,

and glucose production in mammals is primarily a

function of the liver. The postnatal induction of

hepatic glycogenolysis and gluconeogenesis and the

interplay of insulin, glucagon, catecholamines, corticosteroids, and other hormones in the regulation of

hepatic glucose metabolism have been reviewed in

detail by others.2,13,15,22-27 It need only be emphasized

here that the brain appears to be the major determinant



Chapter 12



593



100

Motor development index



105



105

Mental development index



110



Hypoglycemia and Brain Injury



100



95



95



90



85



90



80



85

0



1

3

7

15

Hypoglycemia (days)



0



1

3

7

15

Hypoglycemia (days)



Figure 12-3 Logarithm of days of recorded hypoglycemia lower than 2.6 mmol/L related to Bayley Mental Development Index and Bayley

Psychomotor Development Index at 18 months (corrected age) in a series of 433 premature infants. Regression slopes and 95% confidence

intervals (dashed lines) are shown adjusted for days of ventilation, sex, social class, birth weight, and fetal growth retardation. Data shown are for

both sexes and all social classes combined and for no ventilation. For infants ventilated for 1 to 6, 7 to 14, or more than 14 days, subtract 5, 10, or

15 points, respectively, for mental development index and 4.5, 9.0, or 13.5 points, respectively, for psychomotor development index. (From Lucas

A, Morley R, Cole TJ: Adverse neurodevelopment outcome of moderate neonatal hypoglycaemia, BMJ 297:1304–1308, 1988.)



of (hepatic) glucose production.28 Thus, glucose

production was measured in a series of infants and

children from 1 to 25 kg in body weight by a continuous 3- to 4-hour infusion of the nonradioactive tracer,

6,6-dideuteroglucose. Glucose production on a body

weight basis was found to be twofold to threefold

greater in newborns than in older patients. The infants

clearly had disproportionately higher rates of glucose

production when compared with adult subjects. This

observation becomes understandable when glucose

production is plotted as a function of estimated brain

weight (Fig. 12-4). The linear relationship suggests that

the disproportionately high rates of glucose production

in the neonatal period relate to the disproportionately

large neonatal brain. Because central nervous system

consumption of glucose accounts for 30% or more of

total hepatic glucose output, at least in the premature

infant, this relationship between glucose production

rate and brain weight seems reasonable.29

The mechanisms by which utilization of glucose by

the brain may regulate hepatic glucose output are

unknown. It is possible that the effect is mediated by

subtle changes in blood glucose levels, acting directly

on pancreatic insulin secretion or on hepatic glucose

output. More provocative is the possibility that

the brain mediates control over hepatic glucose

production by neural or hormonal effectors, originating

within the central nervous system.28 This possibility

leads to the interesting logical extension that disturbances of brain may lead to disturbances in glucose

output by liver and result in hypoglycemia or hyperglycemia (see later discussion). Moreover, size of brain



per se may also possibly lead to disturbances in glucose output, secondary to changes in glucose utilization. At any rate, in the normal human, from the

newborn period to adulthood, it is now clear a very

close relationship exists between brain mass and glucose

production.

Glucose Metabolism in Brain

Glucose metabolism in brain is depicted in a simplified

fashion in Figure 12-5. Those aspects particularly relevant to this chapter are shown; further review of cerebral glucose and energy metabolism is contained in

Chapter 6.30-37

Glucose Uptake

Glucose uptake from blood into brain occurs by a

process that is not energy dependent but that proceeds faster than expected by simple diffusion (i.e., carrier-mediated, facilitated diffusion). The transport is

mediated by a specific protein, a glucose transporter.34,35,38-42 The brain glucose transporter is concentrated in capillaries, and the concentration of the

transporter increases with development. In the rat,

the lower apparent blood-brain glucose permeability

in the newborn (%25% of adult values) is related to a

lower concentration of the glucose transporter (not to a

lower affinity of the transporter for glucose). Studies of

human premature infants also suggest that the number

of available endothelial transporters is approximately

one third to one half the value for human adult

brain.29 The importance of the transporter for brain



594



UNIT V



METABOLIC ENCEPHALOPATHIES



240



Glucose production (mg/min)



200



160



120



r = 0.94

80



40



r = 0.95



500

1000

Estimated brain weight (g)



1500



Figure 12-4 Linear relationship between glucose production and

(estimated) brain weight in subjects ranging from premature infants

of approximately 1000 g to adults. Glucose production was measured

by continuous infusion of 6,6-dideuteroglucose. The linear and quadratic functions are depicted by solid and dashed lines, respectively.

(From Bier DM, Leake RD, Haymond MW: Measurement of true glucose

production rates in infancy and childhood with 6,6-dideuteroglucose,

Diabetes 26:1016–1023, 1977.)



function and structure is illustrated by the occurrence

of seizures and developmental delay in infants with partial deficiency of the transporter (see Chapters 5 and

16).39 The glucose concentration normally present in

blood in the newborn rat is approximately one fourth

that required for glucose uptake to proceed at maximal

velocity.34,43 Studies of the human premature infant by positron emission tomography indicate that at a plasma glucose

level of approximately 3mol/mL (i.e., %54 mg/dL), transport

becomes limiting for cerebral glucose utilization.29 Thus,

uptake is one potential site for regulation of glucose metabolism

in brain, and this regulation is particularly dependent on

changes in blood glucose concentrations.

Hexokinase

The initial step in glucose utilization in brain is phosphorylation to glucose-6-phosphate by hexokinase (see

Fig. 12-5). This enzyme is inhibited not only by its

product but also by adenosine triphosphate (ATP).

Under certain circumstances, hexokinase is an important control point in glycolysis.37,39,44,45



Major Fates of Glucose-6-Phosphate

The product of the hexokinase reaction, glucose-6phosphate, is at an important branch point in glucose

metabolism (see Fig. 12-5). From glucose-6-phosphate

originate pathways to the formation of glycogen, to the

pentose monophosphate shunt, and through glycolysis

to pyruvate. Glycogen is important as a readily available

store of glucose in brain; glycogenolysis is an actively

regulated process that is called into play during periods

of glucose lack (i.e., hypoglycemia) or accelerated glucose utilization (e.g., oxygen deprivation [with associated anaerobic glycolysis] or seizures). Glycogen is

concentrated in astrocytes, and with low brain glucose,

astrocytic glycogenolysis is activated to produce glucose-6-phosphate. The latter is converted to lactate,

which then enters the neuron for use as an energy

source (see later).45a The pentose monophosphate shunt

provides reducing equivalents, important for lipid synthesis, and ribose units, important for nucleic acid

synthesis. These two synthetic processes are of particular importance in developing brain. The generation of

reducing equivalents also is critical for generation of

reduced glutathione, crucial for defense against free

radicals and thereby hypoglycemic cellular injury (see

later discussion).

The major fate of glucose-6-phosphate in brain is

entrance into the glycolytic pathway, principally for the

ultimate production of chemical energy in the form of

high-energy phosphate bonds (i.e., ATP and its storage

form, phosphocreatine). When oxidized aerobically,

each molecule of glucose generates 38 molecules of

high-energy phosphate compounds. The next several

sections describe the utilization of glucose for energy

production.

Phosphofructokinase

The most critical step in the glycolytic pathway is

the conversion of fructose-6-phosphate to fructose1,6-diphosphate; the enzyme involved, phosphofructokinase, is a major regulatory, rate-limiting step in

glycolysis (see Fig. 12-5). The enzyme is inhibited by

ATP and is activated by adenosine diphosphate (ADP).

The ammonium ion (NH4+), generated by amino

acid transamination, is also a potent activator of

this complex.

Pyruvate

The glycolytic pathway ultimately results in the

formation of pyruvate, most of which enters the

mitochondrion and is converted to acetyl-coenzyme

A (acetyl-CoA) (see Fig. 12-5). However, pyruvate also

can result in formation of lactate when the cytosolic

redox state is shifted toward reduction. Conversely,

under the conditions of hypoglycemia (i.e., [1]

available lactate and deficient pyruvate, [2] a cytosolic

redox state that is normal or shifted toward oxidation,

and [3] the action of lactate dehydrogenase), lactate can

lead to formation of pyruvate and can become

an energy source (see later discussion). Finally, alanine may be converted to pyruvate by transamination and can therefore become a source of glucose

or acetyl-CoA.



Chapter 12



Hypoglycemia and Brain Injury



595



Glucose (blood)

Uptake by facilitated diffusion

Glucose (brain)

Hexokinase

Pentose

monophosphate

shunt



Glycogen



Glucose-6-phosphate



Fructose-6-phosphate

Phosphofructokinase



Alanine



Pyruvate



Lactate

Fatty acids, cholesterol



Ketone bodies



Acetyl-CoA

Acetylcholine



Aspartate



Oxaloacetate

Citric acid

cycle

␣-Ketoglutarate



Glutamate



CO2

O2

Figure 12-5



ADP ATP



Glutamine ␥-Aminobutyrate

(GABA)



Glucose metabolism in brain. See text for details. ADP, adenosine diphosphate; ATP, adenosine triphosphate.



Acetyl-Coenzyme A

The formation of acetyl-CoA by pyruvate dehydrogenase is the major starting point for the citric acid cycle

(see Fig. 12-5). This step is an important rate-limiting

process in glucose utilization in neonatal brain.34,35

Acetyl-CoA is also the major starting point for the synthesis of brain lipids and acetylcholine. Moreover,

ketone bodies are converted to acetyl-CoA to become

an energy source.

Citric Acid Cycle

The citric acid cycle (with the linked electron transport system) ultimately results in the complete oxidation of the carbon of glucose to carbon dioxide and

the generation of nearly all the ATP derived from this

sugar (see Fig. 12-5). Transamination reactions interface this segment of glucose utilization with certain

amino acids, which thereby can be used for energy

production.

Glucose as the Primary Metabolic

Fuel for Brain

The role of glucose as the primary fuel for the production of chemical energy and the maintenance of normal

function in mature brain are supported by three main

facts.30,34,35,46 First, the respiratory quotient (i.e.,

carbon dioxide output/oxygen uptake) of brain is

approximately 1, a finding indicating that carbohydrate

is the major substrate oxidized by neural tissue.



Glucose is the only carbohydrate extracted by brain in

any significant quantity. Second, cerebral glucose

uptake is almost completely accounted for by cerebral

oxygen uptake. Third, central nervous system function

is rapidly and seriously disturbed by hypoglycemia.

Current data support a similar preeminence for glucose in immature brain.30,35,36 Thus, studies in the newborn dog indicated that glucose consumption in brain

accounts for 95% of cerebral energy supply.47 Moreover,

studies in term fetal sheep demonstrated that, under aerobic conditions, glucose is the main substrate metabolized for energy production.48 Glucose/oxygen quotients

of approximately 1.1 were obtained in two different

laboratories.48-50 (The glucose/oxygen quotient is

equivalent to the arteriovenous difference of glucose

[Â 6] divided by the arteriovenous difference of oxygen

and represents the fraction of cerebral oxygen consumption required for the aerobic metabolism of cerebral glucose.) Although the data demonstrated that glucose is

the primary substrate metabolized by brain, the finding

that the values for glucose/oxygen quotients are slightly

but consistently in excess of 1 suggests that a portion of

the glucose is used for purposes other than complete

oxidation to generate high-energy phosphate bonds.

Other data, based on the fate of labeled glucose in

brain, indicate that glucose is also used for the synthesis

of other materials (e.g., amino acids via transaminations

and lipids via appropriate biosynthetic pathways; see Fig.

12-5).35,46 Syntheses of membrane lipids and proteins,

of course, are critical events in developing brain and



596



UNIT V



METABOLIC ENCEPHALOPATHIES



probably account for a relatively larger proportion of

cerebral glucose utilization than in mature brain.

Important regional and developmental changes in cerebral glucose utilization have been defined, primarily in

animals, but also in human infants.29,30,34-36,51-53 Thus,

early in development, regional differences are relatively

few, and brain stem structures generally exhibit the

highest rates of glucose utilization. With development,

increases in cerebral glucose utilization are most prominent, particularly in cerebral cortical regions. In the

human infant, the developmental progression in the

first year of life occurs first in sensorimotor cortex and

thalamus, next in parietal, temporal, and occipital cortices, and last in frontal cortex and association areas.51,52

Careful studies in animals, focused primarily on electrophysiological maturation of brain stem and diencephalic

structures, showed a close correlation between increases

in rates of glucose utilization and acquisition of neuronal function.34

Alternative Substrates for Glucose in Brain

Metabolism

Overview

Although glucose is the primary metabolic fuel for

brain, it is apparent that certain other substrates also

can be used for energy production and other metabolic

purposes. Under normal circumstances, such alternative substrates are probably not of major importance

for energy production. However, under conditions in

which glucose is limited (e.g., hypoglycemia), alternative

substrates may spare brain function and structure.

Substances such as lactate, pyruvate, free fatty acids,

glycerol, a variety of ketoacids (i.e., ketone bodies), and

certain amino acids have been shown to be capable

of partially or wholly supporting respiration of brain

tissue slices and related in vitro systems.16,33-37,54,55

Certain of these substrates are produced in brain

β-Hydroxybutyrate

β-Hydroxybutyrate

dehydrogenase

Acetoacetate

3-Ketoacid CoAtransferase

Acetoacetyl-CoA

Acetoacetyl-CoA

thiolase

Acetyl-CoA



Amino acids



ATP



Lipids



Figure 12-6 Ketone body use in brain. See text for details. ATP,

adenosine triphosphate; CoA, coenzyme A.



during hypoglycemia (e.g., amino acids from degradation of protein and fatty acids from degradation of phospholipid) and are potentially utilizable as alternative

energy sources (see later discussion). Clearly, however,

these latter alternative substrates are not optimal

because their sources (i.e., proteins and phospholipids)

are largely structural components, and conservation of

energy production at the cost of brain structure is not a

desirable adaptive response. Moreover, because most of

the systemically produced alternative substrates noted

either do not appear in appreciable quantities in blood

or are not capable of crossing the blood-brain barrier to

a major extent, they can contribute relatively little to

brain energy levels in hypoglycemia. The two substrates

most often considered to be useful as primarily bloodborne, alternative sources of brain energy with hypoglycemia are ketone bodies and lactate, and considerable

data show evidence of their value for support of oxidative metabolism in the neonatal brain.16,30,33-36,56-64

Ketone Bodies

Appreciable data have accumulated to suggest that

ketone bodies may be used as alternative substrates

for brain metabolism in the neonatal period. Ketone

bodies are taken up by brain by a carrier-mediated

transport system and are subsequently used according

to the reactions outlined in Figure 12-6.34,65,66

Energy Production. Studies of newborn infants demonstrated that the cerebral extraction of ketone bodies

from blood is markedly greater than in older infants

and adults.34,67 Associated with this finding is an

enhanced rate of ketone body utilization in newborn

brain. Thus, it was shown that ketone bodies accounted for approximately 12% of total cerebral

oxygen consumption in newborns subjected to

6-hour fasts. An enhanced capacity to use ketone

bodies was also demonstrated in human fetal brain.68

These data indicate relevance for animal studies that

demonstrate relatively high activities for the enzymes

involved in ketone body utilization in the immature

versus the mature brain.69-71 These enzymatic activities

also have been demonstrated in the human fetal

brain.72

Thus, the newborn brain, at least under conditions

of brief fasting, normally satisfies a small portion of its

energy demands by the conversion of ketone bodies to

acetyl-CoA, which then proceeds through the citric

acid cycle (see Fig. 12-5). Whether ketone bodies satisfy

a greater portion of cerebral energy demands when glucose is deficient is a separate issue and not so readily

demonstrated (see later). Available data in the newborn

dog do not support an important role for ketone bodies

in this context (see later discussion).30,35,47

Limitations of Hepatic Ketone Synthesis.

Utilization of ketone bodies as alternative substrates for

glucose in brain energy production, under conditions

of glucose deprivation, depends on the capacity of liver

to deliver these compounds to the blood. Data

obtained in human newborn infants suggest that hepatic ketone synthesis is restricted during the early



Chapter 12

TABLE 12-2



Failure of Ketone Bodies to Increase

in Blood with Hypoglycemia

KETONE BODIES (mmol/L)

BetaHydroxybutyrate



0.06 ± 0.01



0.16 ± 0.03

0.24 ± 0.07



Normoglycemic,

term, AGA

Hypoglycemic,

term, AGA

Hypoglycemic, SGA



Acetoacetate



0.31 ± 0.04



Infants



0.02 ± 0.01

0.03 ± 0.01



AGA, appropriate for gestational age; SGA, small for gestational age.

Data from Stanley CA, Anday EK, Baker L, Delivoria-Papadopoulos M:

Metabolic fuel and hormone responses to fasting in newborn

infants, Pediatrics 64:613–619, 1979.



neonatal period.73 The findings demonstrate (1) low

levels of ketone bodies, (2) failure of ketone bodies to

rise with fasting (in contrast to fasting in older children), and (3) failure of ketone bodies to rise with hypoglycemia (Table 12-2). In a subsequent study, relatively

low plasma concentrations of ketone bodies were also

documented with formula feeding.74 Because cerebral

utilization of ketone bodies linearly depends on plasma

concentrations,68 these data from studies of human

infants suggest that limitations of hepatic ketone synthesis

prevent a major role for these materials as alternative

metabolic substrates in brain of human infants with

hypoglycemia. However, these data do not rule out the possibility that exogenous administration of ketone bodies or of

exogenous sources of ketone bodies (e.g., fatty acids) could

serve as alternative metabolic substrates. One report

demonstrated cerebral uptake of exogenously administered beta-hydroxybutyrate for the management of hypoglycemic infants in the first year of life.75

Lactate

Lactate as an important energy source in neonatal

hypoglycemia was suggested by elegant experiments

in the newborn dog.30,34,35,47,63,76,77 Thus, determinations of cerebral metabolic rates for oxygen, glucose,

lactate, and beta-hydroxybutyrate were accomplished

by measurements of CBF and cerebral arteriovenous

differences of these compounds.47 These data then

TABLE 12-3



Lactate as Important Alternative

Substrate for Brain Energy

Production with Hypoglycemia in the

Newborn Dog

SOURCE OF CEREBRAL ENERGY

REQUIREMENTS



Blood Glucose* Glucose Lactate Beta-Hydroxybutyrate

Normoglycemia

Hypoglycemia

(13 mg/dL)

Hypoglycemia

(5 mg/dL)



95%



4%



<1%



48%



52%

56%



2%



597



were used to determine the relative proportions of cerebral energy requirements derived from glucose, lactate,

and beta-hydroxybutyrate under conditions of normoglycemia and insulin-induced hypoglycemia (Table

12-3). During normoglycemia, the newborn dog

obtained 95% of its cerebral energy requirements

from glucose and only a small fraction from lactate

(4%) and beta-hydroxybutyrate (<1%).47 With hypoglycemia, in concert with the expected decline in cerebral utilization of glucose, a striking increase in lactate

use was observed (see Table 12-3). (No appreciable

change in the contribution of ketone body utilization

was noted.) In subsequent experiments, no significant

decrease in brain high-energy phosphate levels

occurred under these conditions.76 Thus, the data indicate that increased utilization of lactate spared brain energy

levels under conditions of severe hypoglycemia.

The mechanisms by which blood lactate leads to

energy production in brain probably include enhanced

lactate uptake by the brain from blood and active oxidation to pyruvate by lactate dehydrogenase (see

Fig. 12-5). Indeed, available data indicate that lactate uptake in newborn dogs occurs at a rate that

exceeds that of adult dogs, even when arterial lactate

concentrations are within or near the physiological

range.30,34,35,47,76,78 Concerning conversion of lactate

to pyruvate, the activity of lactate dehydrogenase in

brain of the perinatal animal has been shown to be

relatively high.34,79-81 Moreover, other data suggest

that neonatal brain may have a particular capability to

use lactate as a brain energy source as an adaptation to

the relative lactic acidemia in the first hours and days

after birth.47,82 Lactic acidemia related to the hypoxic

stress of ‘‘normal’’ vaginal delivery has been documented in newborn rats and lambs.30,34,83,84 These data

also bear on the relative resistance of neonatal versus

adult brain to hypoglycemic injury (see later). The sparing role of lactate in neonatal hypoglycemia requires

further elucidation, but the data from studies of the

newborn dog suggest that this role is considerable.

BIOCHEMICAL ASPECTS OF HYPOGLYCEMIA

The pathophysiological aspects of the encephalopathy

caused by hypoglycemia are best considered in terms

of the initial biochemical effects on brain metabolism,

the later effects, and the combined effects of hypoglycemia with hypoxemia, ischemia, or seizures. These combined effects may be of major clinical relevance because

hypoglycemia uncommonly occurs as an isolated neonatal event and because hypoglycemia not severe

enough to cause brain injury alone may attain that

capacity when combined with certain other deleterious

insults to brain metabolism.



<1%



42%



Hypoglycemia and Brain Injury



*Two hours after injection of insulin (or placebo).

Data from Hernandez MJ, Vannucci RC, Salcedo A, Brennan RW:

Cerebral blood flow and metabolism during hypoglycemia in newborn dogs, J Neurochem 35:622–628, 1980.



Major Initial Biochemical Effects of

Hypoglycemia on Brain Metabolism

Major Biochemical Changes

At the outset, it is crucial to recognize that no biochemical effects of hypoglycemia occur as long as the initial



Cerebral blood flow (mL/100 g/min)



598



UNIT V



METABOLIC ENCEPHALOPATHIES



25

20

15

10

5

0



0-10



10-20 20-30 30-40 40-50 50-60

Blood glucose (mg/dL)



>60



Figure 12-7 Cerebral blood flow as a function of blood glucose measured 2 hours after birth in 25 premature newborns. Cerebral blood

flow was determined by xenon clearance. Note the increase in cerebral

blood flow that begins with blood glucose lower than 30 mg/dL.

(Values are means calculated and redrawn from data from Pryds O,

Christensen NJ, Friis HB: Increased cerebral blood flow and plasma

epinephrine in hypoglycemic, preterm neonates, Pediatrics 85:172–

176, 1990.)



physiological response of increased CBF supplies sufficient glucose to brain. This initial hyperemic response,

first described in adult models of hypoglycemia,33 was

documented in neonatal animal models85,86 and in

human infants.19,87-89 The marked increase in CBF

begins in the human infant when blood glucose declines

to less than approximately 30 mg/dL (Fig. 12-7). Studies

of changes in cerebral blood volume, measured continuously in preterm infants by near-infrared spectroscopy (see Chapter 4), suggested that previously

unperfused capillaries are recruited to maintain glucose

levels in brain with hypoglycemia.89 However, clearly

with marked decreases in cerebral glucose delivery

(e.g., because of marked decreases in blood glucose or

impaired CBF, or both) or with marked increases in

cerebral glucose utilization (e.g., because of seizure),

biochemical derangements begin.

The major initial biochemical effects of hypoglycemia on brain metabolism are summarized in Table

12-4.30,33,35-37,46,47,64,76,90-103 The principal consequences involve cerebral glucose metabolism and the

metabolic attempts to preserve cerebral energy status

by utilization of alternatives to glucose. Sharp falls



TABLE 12-4



Major Initial Biochemical Effects of

Hypoglycemia on Brain Metabolism



## Brain glucose

" Glycogen ! glucose

## CMR glucose

±# CMR oxygen

±# ATP, phosphocreatine

" CMR lactate

# Amino acids, " ammonia

?" Ketone body utilization

# Synthesis of acetylcholine

#, decreased; ##, moderately decreased; ", increased; !, conversion

to; ±, with or without; ATP, adenosine triphosphate; CMR, cerebral

metabolic rate.



in brain glucose concentrations are expected. Glycogenolysis responds in an attempt to restore some of

the brain’s supply of glucose. Nevertheless, the result is

a sharp decrease in the cerebral metabolic rate for glucose. However, an important feature of hypoglycemia,

noted in 1948 in humans,104 and subsequently studied in more detail in experimental animals,30,94,99 is a

disproportionately smaller disturbance of the cerebral metabolic rate for oxygen. Indeed, in neonatal

animals, cerebral metabolic rates of oxygen tend to be

unchanged.30,47 This discrepancy between cerebral utilization of glucose and that of oxygen implies that brain

energy needs are being met by alternative substrates to

glucose (see next paragraph). Indeed, except in very

severe hypoglycemia, significant declines in highenergy phosphate levels in brain are not consistent initial features.

The preservation of oxidative metabolism and highenergy phosphate levels in brain despite the decrease in

cerebral glucose metabolic rate presumably relates to

the utilization of alternative substrates (see Table

12-4). The major substrates considered are lactate,

ketone bodies, and amino acids. As noted previously,

lactate is the most likely candidate as the major alternative substrate for maintenance of brain oxidative

metabolism during hypoglycemia, and the increase in

its rate of utilization in the newborn dog with hypoglycemia is sufficient to account for the preservation of

cerebral metabolic rate for oxygen and of tissue levels

of high-energy phosphate compounds (see Table 123). The possibility of increased ketone body utilization as

an additional alternative energy source is suggested by

data in adult and young (not newborn) animals.99,105

However, as described earlier, direct measurements of

ketone body utilization in the hypoglycemic newborn

dog did not suggest that ketone bodies are important

alternative substrates, at least in that insulin-induced

model.30,34,47 Other alternative substrates for glucose

or energy production, or both, are amino acids. Indeed,

a sharp decrease in brain concentrations of most,

although not all, amino acids occurs, with a consequent increase in brain ammonia levels (through transamination and deamination reactions; see Table 12-4

and subsequent discussion).

Dissociation of Impaired Brain Function and

Energy Metabolism

Changes in brain energy metabolism do not clearly

explain the striking changes in clinical signs and electroencephalographic (EEG) activity of brain during the

initial phases of hypoglycemia. Thus, although findings

differ qualitatively if newborn or adult animals are studied, in general the evolution with hypoglycemia of

clinical changes from an alert state to a depressed

level of consciousness (and even to seizures) and

of EEG changes from normal activity to slowing (and

even to burst-suppression patterns and seizure

discharges) occurs with no definite change in ATP

levels in whole brain or in cerebral cortex and several

other brain regions.30,33,35,46,76,90,98,99 In many

respects, this dissociative phenomenon is similar to

that observed in the initial phases of hypoxic-ischemic



Chapter 12



Hypoglycemia and Brain Injury



599



Major Later Biochemical Effects of

Hypoglycemia on Brain Metabolism



following discussion I review the major effects on the

mature and immature nervous systems separately.



### Brain glucose

### CMR glucose

# CMR oxygen

# ATP, phosphocreatine

" CMR lactate

## Amino acids (except glutamate and aspartate),

" ammonia

# Phospholipids, " free fatty acids

" Intracellular calcium, " extracellular potassium

" Extracellular glutamate

# Glutathione, " oxidative stress



Biochemical Changes in the Mature Animal

Glucose and Energy Metabolism. The biochemical

effects of severe or prolonged hypoglycemia include

an accentuation of the changes described in the previous section as well as the addition of other effects

(Table 12-5). Thus, the decreases in brain glucose

level become very marked, the cerebral metabolic rate

of glucose falls drastically, and a distinct decrease in

cerebral oxidative metabolism and in synthesis of

high-energy phosphate compounds becomes apparent.33,35-37,94,95,97-99,107



TABLE 12-5



#, decreased; ##, moderately decreased; ###, severely decreased; ",

increased; ATP, adenosine triphosphate; CMR, cerebral metabolic

rate.



insults (see Chapter 6). It is unclear whether this occurrence is an adaptive phenomenon (i.e., when faced with

an imminent power failure, the brain curtails neuronal

activity), perhaps to conserve energy stores.

The mechanism by which the dissociation of functional and electrical activity of brain from brain

energy levels occurs may relate to certain of the metabolic concomitants of hypoglycemia (see Table 12-4).

Such concomitants could include alterations in relative

amounts of excitatory and inhibitory amino acids, in

tissue levels of ammonia, or in neurotransmitter

metabolism. Indeed, the degradation of amino acids

described earlier results in an increase in levels of

brain ammonia that are considered potentially sufficient

to produce stupor in adult hypoglycemic animals.99

Whether such levels are generated in newborn animals

is unknown. Data in mature rats rendered hypoglycemic demonstrated that impaired synthesis of acetylcholine

(see Table 12-4) occurs in the first minutes after onset

of hypoglycemia.93,106 Indeed, only modest decreases

in plasma glucose levels caused 20% to 45% decreases

in concentrations of acetylcholine and 40% to 60%

decreases in synthesis of this neurotransmitter in

cortex and striatum.106 The likely mechanism of this

disturbance relates to a decrease in synthesis of

acetyl-CoA because of the drastic decrease in brain glucose and, as a consequence, glycolysis. Even with

modest hypoglycemia, pyruvate concentrations in

brain decrease by 50% in minutes.99 Data concerning

acetylcholine synthesis in hypoglycemic newborn animals are lacking, but similar sharp decreases in brain

glucose and pyruvate35,76 levels suggest that the same

impairment of acetylcholine synthesis is likely.

Major Later Biochemical Effects of

Hypoglycemia on Brain Metabolism

Glucose and Energy Metabolism

The major later biochemical effects of hypoglycemia are

summarized in Table 12-5. Because the largest amount

of available data has been derived from studies of

mature animals, and because the limited data in newborn animals suggest some differences (although many

similarities) compared with mature animals, in the



Metabolic Responses to Preserve Brain Energy

Levels. To preserve brain energy levels, utilization of

endogenous amino acids, derived from protein degradation, glycolytic intermediates, and lactate, continues

as described earlier for initial biochemical effects (glycogen is essentially exhausted by this time). Indeed, as a

consequence of the utilization of amino acids, ammonia levels in the brain increase markedly (i.e., 10-fold to

15-fold). An additional metabolic response (i.e., phospholipid degradation with the generation of free fatty

acids) becomes apparent. The free fatty acids become

an energy source in severe hypoglycemia. However, the

responses are insufficient in severe and prolonged

hypoglycemia to prevent the onset of declines in

levels of high-energy phosphate compounds in brain

and the occurrences of coma and an isoelectric EEG

pattern.33,99 At this point, an additional series of events

develops.

Intracellular Calcium and Cell Injury with

Hypoglycemia. At approximately the time of onset of

EEG isoelectricity, striking changes in intracellular calcium (Ca2+) and extracellular potassium (K+) occur and

appear to initiate a series of events that result in cell

death.33,35-37,97,107 Thus, at this time, the capacity of

the neuron to maintain energy-dependent normal ionic

gradients is lost, extracellular Ca2+ levels decrease

abruptly by approximately sixfold, and extracellular K+

levels increase by approximately 14-fold. Movements of

Ca2+ into the cell and of K+ out of the cell account for

these observations. The initiating event is probably failure of the energy-dependent sodium-K+ (Na+/K+)

pump, which extrudes Na+ and retains K+. With failure

of this system, sodium accumulates intracellularly, K+

is extruded, and sustained membrane depolarization

occurs; the intracellular increase of Na+ then leads to

activation of the Na+/Ca2+ exchange system and movement of Ca2+ intracellularly in exchange for Na+.

Additional crucial effects of this membrane depolarization are excessive release of excitatory amino acids

from synaptic nerve endings and reduced reuptake

secondary to failure of glutamate transport; the

resulting extracellular accumulation of these excitatory neurotransmitters and consequent activation of

glutamate receptors result in a variety of deleterious effects, including influx of Ca2+ (see later).

Ca2+ also may accumulate intracellularly because of



600



UNIT V



TABLE 12-6



METABOLIC ENCEPHALOPATHIES



Cerebral Glucose, Pyruvate, Lactate, and High-Energy Phosphates with Hypoglycemia in the

Newborn Dog

CEREBRAL METABOLITE (PERCENTAGE OF CONTROL){



Blood Glucose (mg/dL)*

20 – 30

10 – 20

<10



Glucose



Pyruvate



Lactate



Phosphocreatine



9%

1%

1%



86%

35%

36%



69%

45%

26%



91%

98%

91%



ATP

93%

100%

97%



*Two hours after insulin injection.

{

All values for glucose, pyruvate, and lactate, but none for phosphocreatine and ATP, were statistically significant from control values.

ATP, adenosine triphosphate.

Data from Vannucci RC, Nardis EE, Vannucci SJ, Campbell PA: Cerebral carbohydrate and energy metabolism during hypoglycemia in newborn dogs,

Am J Physiol 240:R192–R199, 1981.



failure of energy-dependent Ca2+ transport mechanisms designed to maintain low cytosolic Ca2+ levels

(see Chapter 6). The metabolic consequences of these

ionic changes appear to be similar to those described

in Chapter 6 concerning the mechanisms of cell

death with oxygen deprivation. The importance of the

cytosolic Ca2+-induced phospholipase activation (see

Chapter 6) is emphasized by the observation of an

abrupt decline in phospholipid concentration in brain

and a further elevation in free fatty acid concentration.

The deleterious effects of arachidonate (e.g., generation

of free radicals and harmful vasoactive compounds) are

summarized in Chapter 6. Thus, the final common

pathway to neuronal injury in hypoglycemia may be

very similar to that in oxygen deprivation and relates

especially to accumulation of cytosolic Ca2+. Moreover,

in hypoglycemia, as in hypoxia-ischemia, massive

depletion of high-energy phosphate compounds does

not appear to be an obligatory event in producing

cell death.

Role for Excitotoxic Amino Acids in Hypoglycemic

Neuronal Death. As discussed in Chapter 6, considerable data indicate that the mechanism of cell death

with hypoxia-ischemia is mediated by the extracellular

accumulation of excitatory amino acids, which are

toxic in high concentrations. It now appears likely that

excitatory amino acids play a major role in mediation of

neuronal death with hypoglycemia. Evidence in support

of this conclusion includes the demonstrations of a

rise in extracellular concentrations of excitatory

amino acids (aspartate and glutamate) in advanced

hypoglycemia and an attenuation of neuronal injury

by simultaneous administration of antagonists of the

N-methyl-D-aspartate (NMDA) type of glutamate

receptor, both in in vivo models and in cultured neurons.33,35,36,95,108-119 These observations suggest that

the Ca2+ accumulation and Ca2+-mediated deleterious

events, noted in the previous section and described in

detail in Chapter 6, including especially the generation

of reactive oxygen and nitrogen species, are intertwined

with and provoked in considerable part by activation of

the NMDA receptor with resulting influx of Ca2+

through the NMDA channel (as well as through voltage-dependent Ca2+ channels). The free radicals generated result in DNA damage and, as a consequence,

the DNA repair enzyme, poly(ADP-ribose) polymerase-1 (PARP). With excessive activation of PARP and,



as a consequence, adenosine depletion, energy failure

and activation of apoptosis occur.119 PARP inhibitors

have been shown to protect neurons from hypoglycemia in in vitro and in vivo experimental models.119

Thus, the data concerning excitotoxicity and prevention thereof raise interesting new therapeutic possibilities for the prevention or amelioration of hypoglycemic

neuronal death, possibilities that exhibit analogies with

potential therapies for ischemic neuronal death (see

Chapter 6).

Biochemical Changes in the Newborn Animal

Similarities and Differences in Changes in

Newborn and Adult Brain. Many of the biochemical effects of hypoglycemia described earlier in

adult brain can be documented in neonatal brain,

such as sharp decreases in levels of glucose, diminished cerebral utilization of glucose, and diminished concentrations of glycolytic intermediates (Table

12-6).30,35,36,47,76,96,101,118 However, certain metabolic differences from the changes observed in adult brain are

prominent (e.g., preservation of phosphocreatine and

ATP levels despite severe decreases in glucose levels

and markedly greater utilization of lactate as an alternative substrate for energy metabolism). The data contained in Table 12-6 show that hypoglycemia severe

enough to deplete brain of glucose almost entirely is

accompanied by some preservation of such glycolytic

intermediates as pyruvate and lactate and, most strikingly, by complete preservation of phosphocreatine

and ATP levels. Indeed, hypoglycemia of comparable

severity in the adult animal causes marked reductions

in the levels of phosphocreatine and ATP in brain.98,99

The relative preservation of the energy status of

brain with severe hypoglycemia in the newborn

animal is accompanied by a similar preservation of

neurological function and electrical activity. Thus, in the

adult rat, insulin-induced hypoglycemia to plasma glucose values of approximately 35 mg/dL resulted in

prominent slowing on the EEG tracing, and plasma

glucose values of approximately 30 mg/dL resulted in

lethargy and markedly slow activity on the EEG tracing.98,99 Prolongation of this degree of hypoglycemia for

approximately 1 hour resulted in coma and an

isoelectric EEG pattern.98,99 These latter states were

attained in less time in the adult animals when plasma

glucose levels were reduced to 10 to 15 mg/dL.98 In

contrast, in the newborn rat rendered hypoglycemic to



TABLE 12-7



Major Reasons for the Relative

Resistance of the Newborn Animal

to Hypoglycemia



Diminished cerebral energy utilization

Increased cerebral blood flow with even moderate

hypoglycemia

Increased cerebral uptake and utilization of lactate

Resistance of the heart to hypoglycemia



a plasma glucose level of approximately 15 mg/dL, no

change in neurological function could be observed over

2 hours.96 In the newborn dog, prominent slow activity

on the EEG tracing was observed only at plasma glucose

levels lower than approximately 20 mg/dL. Moreover, at

plasma glucose values of approximately 10 to 15 mg/dL

(i.e., levels sufficient to cause an isoelectric EEG pattern

in the adult dog), considerable electrical activity, albeit

slow, was apparent in the newborn dog.76 Indeed, at this

level, seizure discharges often became apparent, and the

accompanying respiratory failure and cardiovascular

collapse could result in death of the animal.76

Reasons for Relative Resistance of Newborn Brain

to Hypoglycemia. The data reviewed demonstrate

clearly a relative resistance of the newborn versus the

adult animal to the deleterious effects of hypoglycemia.

The major reasons for this relative resistance are shown

in Table 12-7. Of particular importance is the lower

cerebral energy requirement in the immature brain

with the consequently lower rate of energy utilization.

This situation, discussed in Chapter 6 concerning the

relative resistance of perinatal brain to hypoxic injury,

presumably relates first to the less-developed dendriticaxonal ramifications and synaptic connections and, as a

consequence, energy-dependent ion pumping and

neurotransmitter synthesis. The second reason for

the relative resistance of the newborn animal and

human to hypoglycemia relates to the marked increase

in CBF, provoked by even moderate hypoglycemia. As

noted earlier, blood glucose levels lower than 30 mg/dL

in the human newborn are associated with prominent

increases in CBF. In mature animals, severe hypoglycemia is required to lead to increases in CBF. The third

reason for the relative resistance to hypoglycemia presumably relates to an increased capacity for both cerebral

uptake and utilization of lactate for brain energy production

(see previous discussion of alternative substrates).30,34,35,47,120 Fourth, severe hypoglycemia does

not have as profound an effect on cardiovascular function in the newborn as in the adult animal.76 The relative resistance of the immature heart relates to its rich

endogenous carbohydrate stores (glucose and glycogen), which can be mobilized for energy during hypoglycemia, and the capacity of the immature heart to use

fuels other than glucose for energy.121-123 Thus,

although it is clear that more data are needed concerning the impact of hypoglycemia on neonatal brain, current information suggests that cerebral and myocardial

metabolic capacities provide remarkable degrees of

resistance.



Glutamate (% of baseline value)



Chapter 12



601



Hypoglycemia and Brain Injury



300

Control

Hypoglycemia

200



100



0

0



30



60



90



120



150



180



210



240



Time (min)

Figure 12-8 Increase in extracellular glutamate with hypoglycemia

in the immature rat. The striatal glutamate efflux (i.e., extracellular

glutamate) in control (n = 6) and hypoglycemic (n = 6) postnatal day 7

rats was determined by microdialysis. Hypoglycemia was produced by

insulin injection. In hypoglycemic animals, the striatal glutamate efflux

increased gradually and peaked at 240% of control values. (From

Silverstein FS, Simpson J, Gordon KE: Hypoglycemia alters striatal

amino acid efflux in perinatal rats: An in vivo microdialysis study, Ann

Neurol 28:516–521, 1990.)



Role for Excitotoxic Amino Acids in Hypoglycemic

Neuronal Death. A possible role for excitatory amino

acids in the hypoglycemic neuronal death with severe

hypoglycemia in neonatal animals, as in mature animals (see earlier discussion), is suggested by studies

of severe insulin-induced hypoglycemia in 7-day-old

rats.124 Thus, insulin-induced hypoglycemia caused

an increase in striatal extracellular glutamate, measured

by microdialysis, with the onset of the increase at

blood glucose levels of 20 mg/dL (Fig. 12-8). After

31=2 hours of hypoglycemia (terminal glucose level of

<5 mg/dL), striatal glutamate was approximately 2.4fold higher than baseline levels. The increased

extracellular glutamate may be caused by failure of

high-affinity glutamate uptake mechanisms or by

increased release (secondary to synaptic release provoked by membrane depolarization [resulting from

Na+ or Ca2+ influx] or by reversal of the Na+-dependent

glutamate transport system [resulting from increased

intracellular Na+], or by both mechanisms). The potential consequence would be excitotoxic neuronal death

by the mechanisms described in Chapter 6. Prevention

of neuronal death in organotypic hippocampal cultures

derived from newborn rat brain and maintained in the

absence of glucose by the NMDA receptor antagonist

MK-801 also illustrates the importance of excitotoxic

mechanisms in hypoglycemic neuronal death.115 The

prevention of neuronal death by addition of the antagonist 30 minutes after the insult may have important

therapeutic implications. The increase in apparent

affinity of the NMDA receptor observed in the hypoglycemic piglet suggests that the excitotoxic potential of

glutamate may be enhanced by hypoglycemia.118



METABOLIC ENCEPHALOPATHIES



Glutathione Depletion and Oxidative Stress. A role

for oxidative stress in hypoglycemic cell death was suggested by studies of cultured neural cells and a newborn piglet model.125,126,126a In cultured cells, glucose

deprivation caused a decrease in glutathione levels and

then cell death. Glucose is involved in production of

reduced glutathione by generating reducing equivalents and by providing a carbon source required for

biosynthesis of this critical antioxidant. That the cell

death in the studies of cultured glial cells was mediated

by oxidative stress and free radical attack was shown by

the demonstration of protection by free radical scavengers. Because Ca2+ influx and glutamate receptor activation in neurons may lead to generation of free

radicals (see Chapter 6), the deleterious effect of reduced glutathione levels could be critical in the final

common pathway to cell death with hypoglycemia.

Studies of the hypoglycemic newborn piglet showed

markedly elevated mitochondrial production of reactive

oxygen species.126 The demonstration that brainderived neurotrophic factor (BDNF) protected cultured

neurons from hypoglycemic injury further suggests a

role for oxidative stress because BDNF induces antioxidant systems.127



Hypoglycemia and Hypoxemia or Asphyxia

Hypoglycemia and Hypoxemia

The vulnerability of immature brain to hypoxemic

injury is enhanced by concomitant hypoglycemia,

an observation first made in 1942.128 Studies of cerebral carbohydrate metabolism during hypoxemia

and hypoglycemia in newborn rats provided further

insight into the mechanism of this effect.30,96 Thus,

newborn rats subjected to hypoxemia by breathing

100% nitrogen exhibited greater mortality rates

when they were also subjected to insulin-induced

hypoglycemia (Fig. 12-9). Indeed, animals rendered hypoglycemic for 1 hour experienced a fivefold reduction

in survival capability, and those hypoglycemic

for 2 hours did even worse. Supplementation of hypoglycemic animals with glucose before anoxia improved

outcome (see Fig. 12-9). Animals rendered hypoglycemic as well as hypoxemic exhibited less accumulation

of lactate in brain and a faster decline in cerebral

energy reserves (ATP and phosphocreatine) than

those rendered hypoxemic alone. Moreover, glucose

supplementation ameliorated the adverse metabolic

effects. The mechanism for the enhanced deleterious

effect of hypoxemia when hypoglycemia was associated

appeared to relate to a diminution in brain glucose

reserves and thus retarded glycolytic flux. The

improvement with glucose supplementation supports

this notion.

Studies of cultured immature glial cells are relevant

to the adverse effect of the combination of hypoxemia

and hypoglycemia. Thus, not only are immature astrocytes more vulnerable to glucose deprivation than are

mature glial cells,129 but also, of special interest in this

context, glucose deprivation markedly accentuates the



100

Hypoglycemia

+

Glucose

10’

30’



80

Survival (%)



UNIT V



Control



60



40



20



60’

120’



0



Hypoglycemia

5



10



15

20

Anoxia (min)



25



30



Figure 12-9 Deleterious effect of hypoglycemia on vulnerability to

anoxia (nitrogen breathing). The percentage of survival of newborn

rats was determined as a function of duration of anoxia.

Hypoglycemia was produced by insulin injection 1 to 2 hours before

the onset of anoxia; some hypoglycemic animals were pretreated with

glucose (1.8 g/kg, subcutaneously) either 10 or 30 minutes before

anoxia. (From Vannucci RC, Vannucci SJ: Cerebral carbohydrate metabolism during hypoglycemia and anoxia in newborn rats, Ann Neurol

4:73–79, 1978.)



vulnerability of differentiating glial cells to oxygen deprivation (Fig. 12-10).130 This effect of glucose deprivation on immature glial cells is apparent in both

differentiating astrocytes and oligodendroglia.130



100



80

LDH efflux (% of total)



602



60



40



20



0



3



5.6

10

15

20

Glucose concentration (mmol/L)



25



Figure 12-10 Beneficial effect of glucose on hypoxia-induced cellular injury in differentiating glial cells, primarily astrocytes. Cellular

injury was determined by measurement of efflux of lactate dehydrogenase (LDH) from the damaged cells. Primary glial cell cultures were

grown for 18 days, when differentiation was active, and subjected to

hypoxia, with the indicated concentrations of glucose in the culture

medium. (From Callahan DJ, Engle MJ, Volpe JJ: Hypoxic injury to developing glial cells: Protective effect of high glucose, Pediatr Res 27:186–

190, 1990.)