— A method has been developed for the simultaneous measurement of the rates of glucose consumption in the various structural and functional components of the brain in vivo. The method can be applied to most laboratory animals in the conscious state. It is based on the use of 2-deoxy-D-[14C]glucose ([14C]DG) as a tracer for the exchange of glucose between plasma and brain and its phosphorylation by hexokinase in the tissues. [14C]DG is used because the label in its product, [14C]deoxyglucose-6-phosphate, is essentially trapped in the tissue over the time course of the measurement. A model has been designed based on the assumptions of a steady state for glucose consumption, a first order equilibration of the free [14C]DG pool in the tissue with the plasma level, and relative rates of phosphorylation of [14C]DG and glucose determined by their relative concentrations in the precursor pools and their respective kinetic constants for the hexokinase reaction. An operational equation based on this model has been derived in terms of determinable variables. A pulse of [14C]DG is administered intravenously and the arterial plasma [14C]DG and glucose concentrations monitored for a preset time between 30 and 45min. At the prescribed time, the head is removed and frozen in liquid N2-chilled Freon XII, and the brain sectioned for autoradiography. Local tissue concentrations of [14C]DG are determined by quantitative autoradiography. Local cerebral glucose consumption is calculated by the equation on the basis of these measured values.

The method has been applied to normal albino rats in the conscious state and under thiopental anesthesia. The results demonstrate that the local rates of glucose consumption in the brain fall into two distinct distributions, one for gray matter and the other for white matter. In the conscious rat the values in the gray matter vary widely from structure to structure (54-197 μmol/100 g/min) with the highest values in structures related to auditory function, e.g. medial geniculate body, superior olive, inferior colliculus, and auditory cortex. The values in white matter are more uniform (i.e. 33–40 μmo1/100 g/min) at levels approximately one-fourth to one-half those of gray matter. Heterogeneous rates of glucose consumption are frequently seen within specific structures, often revealing a pattern of cytoarchitecture. Thiopental anesthesia markedly depresses the rates of glucose utilization throughout the brain, particularly in gray matter, and metabolic rate throughout gray matter becomes more uniform at a lower level.

The [14C]deoxyglucose method for the measurement of local cerebral glucose utilization: theory, procedure, and normal values in the conscious and anesthetized albino rat.

Light microscopy immunohistochemical techniques were used to examine the distribution of glutamine synthetase in rat brain. Glutamine synthetase was found to be localized in the glial cells. Neuronal cell bodies, endothelial cells, and choroid epithelium contained no enzyme. The findings indicate that glia have a crucial role in glutamic acid, gamma-aminobutyric acid, and ammonia metabolism in brain.

https://science.sciencemag.org/content/sci/195/4284/1356.full.pdf

Glutamine synthetase: glial localization in brain.

A method has been developed to measure local glucose consumption in the various structures of the brain in man with three-dimensional resolution. [18F]-2-deoxy-2-fluoro-D-glucose is used as a tracer for the exchange of glucose between plasma and brain and its phosphorylation by hexokinase in the tissue. A mathematical model and derived operational equation are used which enable local cerebral glucose consumption to be calculated in terms of the following measurable variables. An intravenous bolus of [18F]-2-deoxy-2-fluoro-D-glucose is given and the arterial specific activity monitored for a predetermined period of from 30 to 120 minutes. Starting at 30 minutes, the activity in a series of sections through the brain is determined with three-dimensional resolution by an emission tomographic scanner. The method was used to measure local cerebral glucose consumption in two normal volunteers. The values in gray matter structures range from 5.79 mg/100 g per minute in the cerebellar cortex to 10.27 in the visual cortex, whereas, in white matter structures, the values range from 3.64 mg/100 g per minute in the corpus callosum to 4.22 in the occipital lobe. Average values for gray matter, white matter, and whole brain metabolic rates, calculated as a weighted average based on the approximate volume of each structure, are 8.05, 3.80, and 5.90 mg/100 g per minute, respectively. The value of 5.9 mg/100 g per minute compares favorably with values previously reported.

The [18F]fluorodeoxyglucose method for the measurement of local cerebral glucose utilization in man.

Astrocytes are the most numerous cell type in the central nervous system. They provide structural, trophic, and metabolic support to neurons and modulate synaptic activity. Accordingly, impairment in these astrocyte functions during brain ischemia and other insults can critically influence neuron survival. Astrocyte functions that are known to influence neuronal survival include glutamate uptake, glutamate release, free radical scavenging, water transport, and the production of cytokines and nitric oxide. Long-term recovery after brain injury, through neurite outgrowth, synaptic plasticity, or neuron regeneration, is influenced by astrocyte surface molecule expression and trophic factor release. In addition, the death or survival of astrocytes themselves may affect the ultimate clinical outcome and rehabilitation through effects on neurogenesis and synaptic reorganization.

Astrocytes and brain injury.

Transport and metabolism of calcium ions in nerve

— The question of a constant density of glial cells in mammalian cerebral cortex regardless of species was examined by surveying the cortical activities of two enzymes primarily localized to dial cells. The cortical activity of butyrylcholinesterase (EC 3.1.1.8) was essentially constant at a rate of approx. 0.1 μmol of butyrylthiocholine hydrolysed min-1 g-1 over the range of species from rat (brain wt., 1.6 g) to fin whale and sperm whale (brain wt., 6800 and 7800 g, respectively). Over the same range the activity of cortical acetylcholinesterase, a neuronal enzyme, decreases by a factor of 7. Thus, butyrylcholinesterase ranged from < 2 per cent (in small rodent brains) to approximately 10 per cent (in whale brain) of the cortical acetylcholinesterase activity. The cortical activity of carbonic anhydrase (EC 4.2.1.1) was constant at a rate of 6.2 (± 0.25) μmol of CO2 evolved min-1 g-1 over the range of species from guinea-pig (brain wt., 4.75 g) to fin whale (brain wt., 6800 g). These data obtained by assaying the dehydration reaction were confirmed by limited assays of the esterase activity of the enzyme (with p-nitrophenylacetate as substrate) and agreed with limited, previously reported data for the hydration reaction. Thus, the circumstantial evidence strongly favoured a relative constancy of cortical glial cell density regardless of species.



The rates of anaerobic glycolysis in the cerebral cortex of various species were also investigated. For six species from mouse (brain wt., 0.4 g) to beef (brain wt., 380 g) cortical anaerobic glycolysis varied only slightly in the range of 50–62 μmol of CO2 evolved h-1 g-l, whereas cortical oxygen consumption for the same range of species decreased by a factor of 3. Previously frozen samples of beef cortex glycolysed at 35 per Cent of the rate of fresh (unfrozen) samples. Since identical rates were obtained for previously frozen samples of fin whale cerebral cortex, we concluded that the relative constancy of cortical anaerobic glycolysis could be extended to the range from mouse to whale and that this aspect of cortical metabolism is probably primarily glial in localization. Some implications of the latter conclusion for the proposed role of astrocytes as modulators of neuronal activity have been discussed.

The activities of butyrylcholinesterase and carbonic anhydrase, the rate of anaerobic glycolysts, and the question of a constant density of glial cells in cerebral cortices of various mammalian species from mouse to whale

PREVIOUS iiivestigations have characterized 2-deoxy-~-glucose* as an inhibitor of glucose metabolism. Its effectiveness in inhibiting glycolysis and growth of neoplastic tissues (ELY, 1954; WOODWARD and HUDSON, 1954; SOKOLOFF et al., 1956; BALL, WICK and SANDERS, 1957) suggested clinical applications. During evaluations of its toxicity in animals preliminary to such trials, LANDAU and LUBS (1958) observed neurological signs and symptoms which closely resembled those of insulin-induced hypoglycaemia. After oral or parenteral administration, 2-deoxyglucose is rapidly distributed throughout the body and appears promptly in the cerebrospinal fluid (WICK, DRURY and MORITA, 1955; LANDAU and LUBS, 1958). Using doses of the order of 12 m-moles/kg, LANDAU and LUBS (1958) found an elevation of blood glucose to levels two or three times normal control values. Under such conditions both blood glucose and 2-deoxyglucose respond to insulin (WICK et al., 1955), but the insulin-mediated decrease in blood glucose to normal ranges intensifies the apparent hypoglycaemic picture. These observations are consistent with inhibition of tissue utilization of glucose in the presence of 2-deoxyglucose, resulting in a situation of simultaneous hyperglycaemia and cytoglycopenia, which in the central nervous system is expressed as dysfunctions usually associated with hypoglycaemia. 2-Deoxyglucose is readily phosphorylated by yeast, muscle and brain hexokindse (SOLS and CRANE, 1954; WOODWARD and HUDSON, 1955; WICK et al., 1957), and it produces inhibition of glycolysis in yeast and various mammalian tissues, including brain (WOODWARD, 1952; WOODWARD and HUDSON, 1954). Metabolism of 2deoxyglucose-6-phosphate appears not to occur (SOLS and CRANE, 1954; WICK et al., 1957) and in purified kidney preparations it has been reported to block conversion of glucose-6-phosphate to fructose-6-phosphate, with possible secondary effects on hexokinase (WICK et al., 1957). Few studies of the effects of 2-deoxyglucose on cerebral metabolism have been reported. Anaerobic glycolysis of rat cerebral cortex slices is inhibited 50 per cent when incubated with 1 m~-2-deoxyglucose plus 15 mwglucose in vitro, a result comparable to those obtained with rat diaphragm or with tumour tissues under similar conditions, but oxygen uptake by rat cerebral cortex appears much less

The effects of 2-deoxy-D-glucose on metabolism of slices of cerebral cortex incubated in vitro.

Effects of NH4Cl on oxidative metabolism of cat cerebral cortex slices and mitochondria incubated in vitro were studied. In slices, addition of 10 mm NH4Cl to the incubation medium resulted in sign...

Ammonia toxicity and cerebral oxidative metabolism.

SUMMARY

The distribution of indicator solutes (inulin, sucrose, raffinose, thiocyanate, chloride, and isethionate) and the swelling in slices of adult cat cerebral cortex incubated in vitro have been investigated for a variety of incubation media and conditions. Where appropriate, these data have been compared with analogous data obtained on cat cerebral cortex in vivo (Bourkeet al., 1965) and with data previously reported for slices of rat cerebral cortex (Pappius and Elliott, 1956; Pappiuset al., 1962) and guinea pig cerebral cortex (Varon and McIlwain, 1961; Keeseyet al., 1965) incubated in vitro under comparable conditions.



In addition to substantial agreement of present data with previously reported in vitro data, a number of new findings have been added by the present study: (a) a component of slice swelling which is K+-dependent; (b) the association of slice swelling with presence of a diffusible, external anion (Cl−) and its prevention by replacement with a relatively non-diffusible anion (isethionate−); (c) variation of the size of slice chloride spaces as a direct function of slice swelling; (d) dependence of the size of slice fluid spaces accessible to inulin and sucrose upon time, during incubation, of solute addition and upon K+ concentration of the incubation medium; (e) indications of the dynamic and presumably metabolically-dependent nature of indicator solute distribution; and (f) the mobility of a portion of the fluid of swelling associated with changing the K+ concentration but not the tonicity of the medium during incubation.



At least five operationally-defined fluid compartments may be inferred from the present data : (1) interstitial or extracellular space(s) readily accessible to all solutes and of a size which can be minimally estimated from direct determinations in viuo; (2) additional fluid space(s) accessible to most solutes, including inulin and sucrose, under apparently suboptimal conditions of slice metabolism in vitro; (3) fluid space(s) prone to swell under in vitro conditions and readily accessible in vitro to chloride and thiocyanate but not to inulin or sucrose; (4) fluid space(s) which swell reversibly in the presence of added external K+ (or Rb+) and are inaccessible to all usual indicator solutes; and (5) after exclusion of the foregoing, the remaining fluid presumably comprising most of the intracellular space(s).



The data have been discussed in terms of the morphological complexity of cerebral cortex, in terms of applicability to studies of cortical electrolyte distribution, and in terms of the general problem of delineating cerebral interstitial spaces.

Fluid compartmentation and electrolytes of cat cerebral cortex in vitro–i swelling and solute distribution in mature cerebral cortex*

Summary The distribution of systemically or locally administered 5-fluorouracil-2-14C (5-FU) in brain and cerebrospinal fluid (CSF) of the monkey (Macaca mulatta) has been investigated. Following controlled i.v. injection, there was rapid penetration of the 5-FU into brain and CSF, concomitant with a rapid loss of 5-FU from the systemic plasma compartment. CSF repeatedly sampled from various sites (lumbar, cisternal, intraventricular, and cortical surface) displayed characteristic and different kinetic profiles (drug concentrations versus time) following initial i.v. injection of 5-FU. Despite the fact that 91% of the injected dose of 5-FU was cleared from the vascular compartment within 5 min after the start of the i.v. injection (5 ml at 1.086 ml/min) and 98% of the injected dose was cleared by 1 hr, approximately 0.126% of the total administered dose represented the average integrated content of the drug in the entire CSF compartment over the experimental hr. Similarly, the average integrated content of the drug in the brain over the experimental hr represented 0.172% of the total administered dose of 5-FU, as determined by sampling cerebral and cerebellar cortex, subcortical cerebral and cerebellar white matter, striatum, pons, and medulla oblongata. Labeled 5-FU gained access to the CSF from the plasma by simple diffusion. The average integrated concentration of 5-FU in CSF over the experimental hr was increased by 48% when the rate of i.v. injection was increased fourfold. As demonstrated by bilateral perfusion over the exposed cerebral cortex or by bilateral ventriculocisternal perfusion, 5-FU in the CSF was an adequate source for delivery of the drug to contiguous brain tissue. Studies of the regional brain contents of 5-FU following combined i.v. and ventriculocisternal administration of the drug are presented, together with investigations of the physicochemical and metabolic characteristics of the drug. Our studies provide in the primate a model for the determination of the kinetics of distribution of antineoplastic agents in brain and CSF following i.v. administration or following perfusion into the CSF pathway.

https://cancerres.aacrjournals.org/content/canres/33/7/1735.full.pdf

Donald B. Tower

Papers

The activities of butyrylcholinesterase and carbonic anhydrase, the rate of anaerobic glycolysts, and the question of a constant density of glial cells in cerebral cortices of various mammalian species from mouse to whale

The effects of 2-deoxy-D-glucose on metabolism of slices of cerebral cortex incubated in vitro.

Ammonia toxicity and cerebral oxidative metabolism.

Fluid compartmentation and electrolytes of cat cerebral cortex in vitro–i swelling and solute distribution in mature cerebral cortex*

Kinetics of Entry and Distribution of 5-Fluorouracil in Cerebrospinal Fluid and Brain following Intravenous Injection in a Primate