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 z -d e o x y -~ ['4C]glucose (['4C]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, [ '4C]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 N,-chilled Freon XII, and the brain sectioned for autoradiography. Local tissue concentrations of ['4C]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 (54-197pmo1/100g/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 pmo1/100 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 MAMMALIAN brain is a complex heterogeneous organ comprising many structural and functional components with different and independently regulated levels of functional activity and energy metabolism. Much of our present knowledge of cerebral .

Summary: A theoretical model of blood -brain exchange is developed and a procedure is derived that can be used for graphing multiple-time tissue uptake data and deter mining whether a unidirectional transfer process was

Summary:The method of graphical analysis for the eval uation of sequential data (e.g., tissue and blood concen trations over time) in which the test substance is irre versibly trapped in the system has been expanded. A sim pler derivation of the original analysis is presented.General equations are derived that can be used to analyze tissue uptake data when the blood-plasma concentration of the test substance cannot be easily measured. In ad dition, general equations are derived for situations when trapping of the test substance is incomplete and for a combination of these two conditions. These derivationsThe study of the movement of solute molecules across tissue capillaries and their localization within selected tissues of both animal and human subjects has been greatly facilitated by the use of tomo graphic machines. These instruments can provide sequential measurements over time of regional tissue concentrations. The general experimental protocol usually involves the injection of the test substance into the blood of the subject and then measuring the concentration of the substance in both the blood and various tissue regions over a period of time. These data may then be analyzed by a number of methods to ascertain the desired parameters of the system.One method of analysis that has been proposed for those substances that are irreversibly trapped in the system is a graphical analysis of multiple time data points . For this method, an equation has been developed for a very general model. When certain measurable quantities are This approach is also shown to result in equations with at least one less nonlinear term than those derived from direct compartmental analysis. Specific applications of these equations are illustrated for a compartmental system with one reversible region (with or without re versible binding) and one irreversible region.

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Studies were carried out in anesthetized rats comparing dynamics and pathways of interstitial fluid (ISF) drainage from different regions of brain. Rates of drainage from brain and flow into cerebrospinal fluid (CSF) were estimated from the efflux from brain and influx into CSF of radioiodinated albumin (RISA) following microinjection into caudate nucleus, internal capsule, or midbrain: pathways of flow through the subarachnoid space and into lymph were traced from the distribution of horseradish peroxidase and/or Evans blue-labeled albumin after injection into brain. ISF drainage rates (mul X g brain-1 X min-1) estimated for the three injection sites were 0.18, 0.19, and 0.29, respectively. Flow of ISF into bulk CSF sampled from the cisterna magna accounted for 60-75% of efflux from midbrain but only 10-15% of efflux from caudate nucleus or internal capsule. RISA was concentrated in the subarachnoid space, relative to bulk CSF, in sleeves of adventitial tissue surrounding pericerebral arteries, possibly accounting for the low recovery of isotope from bulk CSF. From the subarachnoid space, some fluid drained via olfactory nerve sheaths to retropharyngeal lymph nodes.

Experiments were carried out to evaluate the role of convection in the removal of large molecules from brain interstitial fluid. Radiolabeled test compounds were injected into the caudate nucleus of anesthetized rats through a guide cannula implanted 1 wk previously and the concentrations of isotope in brain and cerebrospinal fluid (CSF) determined at various times after injection. Control studies with 22Na indicate that the permeability of the blood-brain barrier is normal in tissue surrounding the intracerebral injection cannula. For 69,000 dalton serum albumin, 4,000 dalton polyethylene glycol, and 900 dalton polyethylene glycol, clearance from brain approximates a single exponential decay with half times of disappearance of 12.2, 12.6, and 14.4 h, respectively. Similarly in efflux rate, despite a fivefold difference in diffusion coefficient, is consistent with convective losses from brain, and the maximal rate of interstitial fluid removal estimated on the basis of these data is 0.11 microliter.g brain-1.min-1. Only 10-20% of total efflux is into bulk CSF withdrawn from the cisterna magna.

The transport of alpha-aminoisobutyric acid (AIB), N-methyl-AIB (MeAIB), and diethylenetriaminepentaacetic acid (DTPA) from blood to brain was measured over different experimental periods in eight regions of the rat brain. Unidirectional transfer rate constants were determined from multiple-time/graphical and single-time analysis of the experimental data; values of 0.0018, 0.00057, and 0.000021 ml g-1 min-1, respectively, were obtained for the thalamus by graphical analysis. The initial distribution volume of AIB and MeAIB in brain tissue was several-fold greater than that of DTPA and the tissue plasma volume, and this difference was not accounted for by red blood cell uptake. This discrepancy could be due to rapid transport of AIB and MeAIB into brain endothelial cells in addition to the relatively rapid uptake by choroidal, meningeal, and ependymal associated tissues that was demonstrated by autoradiography. Thus, it may be misleading and erroneous to consider the blood-brain barrier (BBB) to be a simple, single-membrane structure when analyzing the blood-brain transfer data of solutes such as amino acids. The data from the ventriculocisternal perfusion experiments and previously published AIB uptake data in mouse brain slices were used to estimate the transfer rate constants across brain cell membranes. These studies indicated that the transport of AIB into brain cells was approximately 110 to 265 times greater than that across normal brain capillaries per unit mass of brain tissue, and that the BBB limits blood-to-brain cell transport of this amino acid. These observations (low rate of transport across normal brain capillaries and rapid concentrative uptake by brain cells) indicate that AIB is a good marker for measuring moderate to large increases in BBB permeability by experiments that require unidirectional flux of the tracer.

The ontogeny of regional blood-brain barrier function was quantified with the rate constant for influx (Ki) across the blood-brain barrier with the small molecular weight synthetic, inert hydrophilic amino acid alpha-aminoisobutyric acid (AIB) in chronically instrumented early (87 days of gestation, 60% of gestation) and late (137 days of gestation, 90% of gestation) gestation fetal, newborn (3 days of age), older (24 days of age), and adult (3 years of age) sheep. The Ki was significantly (P < 0.05) lower in the brain regions of the adult sheep and in most brain regions of newborn and older lambs compared with fetuses at 60 and 90% of gestation. The Ki exhibited regional brain heterogeneity (P < 0.05) in the five groups. The patterns of regional heterogeneity were accentuated (P < 0.05) in the younger groups. We conclude that ontogenic decreases in blood-brain barrier permeability are observed in ovine fetuses from 60% of gestation to maturity in the adult.