Karen D. Pettigrew

THE [14C]DEOXYGLUCOSE METHOD FOR THE MEASUREMENT OF LOCAL CEREBRAL GLUCOSE UTILIZATION: THEORY, PROCEDURE, AND NORMAL VALUES IN THE CONSCIOUS AND ANESTHETIZED ALBINO RAT

Abstract— 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.

Hypoxia Increases Velocity of Blood Flow through Parenchymal Microvascular Systems in Rat Brain

The postulation that hypoxia increases local cerebral blood flow (lCBF) mainly by perfusing more capillaries (the capillary recruitment hypothesis) was tested in awake adult male Sprague–Dawley rats exposed to 10% O2 and control rats. The [14C]iodoantipyrine technique was used to measure lCBF. Local cerebral blood volume was determined by measuring plasma and red cell distribution spaces within the brain parenchyma with 125I-labeled serum albumin (RISA) and 55Fe-labeled red cells (RBC), respectively. Tissue radioactivity in 44 brain areas was estimated by quantitative autoradiography. Hypoxia raised lCBF by 25–90% in all brain areas. In about one-quarter of the brain areas, the rise in blood flow was associated with a small increase in microvascular plasma and blood volumes. This change in blood volume, which could be the result of perfusing more parenchymal microvessels and/or increasing parenchymal microvessel diameter, is not sufficient to account for the observed rise in lCBF. In the remaining areas the RISA, RBC, and blood spaces were either unchanged or only marginally increased by hypoxia. For this hypoxic perturbation, the major mechanism of raising blood flow appears to be increased velocity of microvessel perfusion and not perfusion of more capillaries. These findings provide only limited support for the capillary recruitment hypothesis.

Early detection of Alzheimer's disease : a statistical approach using positron emission tomographic data

Correlational analysis of regional cerebral glucose metabolism (rCMRglc) obtained by high-resolution positron emission tomography (PET) has demonstrated reduced neocortical rCMRglc interactions in mildly/moderately demented patients with probable Alzheimers disease (AD). Thus, identification of individual differences in patterns of rCMRglc interactions may be important for the early detection of AD, particularly among individuals at greater risk for developing AD (e.g., those with a family history of AD). Recently, a statistical procedure, using multiple regression and discriminant analysis, was developed to assess individual differences in patterns of rCMRglc interdependencies. We applied this new statistical procedure to resting rCMRglc PET data from mildly/moderately demented patients with probable AD and age/sex-matched controls. The aims of the study were to identify a discriminant function that would (a) distinguish patients from controls and (b) identify an AD pattern in an individual at risk for AD with isolated memory impairment whose initial PET scan showed minor abnormalities, but whose second scan showed parietal hypometabolism, coincident with further cognitive decline. Two discriminant functions, reflecting interactions involving regions most involved in reduced correlations in probable AD, correctly classified 87% of the patients and controls, and successfully identified the first scan of the at-risk individual as AD (probability >0.70). The results suggest that this statistical approach may be useful for the early detection of AD.

Regional cerebrovascular permeability to [14C]sucrose after osmotic opening of the blood-brain barrier.

Recent studies demonstrate that infusion of a hypertonic solution of a watersoluble non-electrolyte into the carotid artery of animals opens the blood-brain barrier, probably by shrinking cerebrovascular endothelial cells and widening interendothelial tight junctions1,12,14. Such osmotic treatment increases the brain :plasma ratio of tracers that are injected intravenously2,12,~4,16, and increases the brain uptake index or indicator dilution extraction of poorly diffusible tracersS,lo,lz, ~5. However, the actual change in cerebrovascular permeability following hypertonic infusion has not been measured directly. Plasma tracer concentrations were not followed in the experiments with intravenous injection, and the brain uptake index or indicator dilution techniques may be inadequate because they can be affected by changes of cerebral blood flow; they apply to whole brain rather than to brain regions, and they are not sensitive enough to measure permeability coefficients less than 10 -6 cm/sec3,4,6,8,1~,12,15. We therefore developed a new method that is not subject to these objections, and which can quantify normal cerebrovascular permeability and changes in permeability caused by osmotic barrier treatment 8. Adult, male, Osborn-Mendel rats were anesthetized with sodium pentobarbital, i.p. Indwelling catheters were placed in the femoral artery and vein and in the right external carotid artery. As has been described, 1.6-1.8 molal arabinose solution in water, or 0.9 ~ (w/v) NaCl, warmed to 37 °C, was infused in a retrograde direction into the external carotid artery for 30 sec at a rate of 0.12 ml/sec. This flow rate washes out blood from the ipsilateral cerebral hemisphereL Two ml/kg of 2 g/100 ml Evans blue (Chroma-Gesellschaft, Stuttgart) in isotonic saline was injected, i.v., 5 min prior to carotid infusion, and was used as a visual indicator of barrier opening 14. Five #Ci of [14C]sucrose (New England Nuclear, Boston;

Reproducibility of Resting Cerebral Blood Flow Measurements with H215O Positron Emission Tomography in Humans

Two consecutive measurements of resting CBF were carried out in normal volunteers (n = 25) using H215O positron emission tomography. Absolute whole-brain blood flow (WBBF; ml 100 g−1 min−1, mean ± SD) for the first (40.3 ± 6.4) and second (39.3 ± 6.5) measurements was not significantly different (mean % difference 2.3 ± 8.7). Analysis of regions of interest showed no significant differences in absolute regional CBF (rCBF) and normalized (rCBF/WBBF) rCBF. Left-right differences were also not significant. These data demonstrate the reproducibility of resting CBF measurements in normal humans.

Mutant herpes simplex virus induced regression of tumors growing in immunocompetent rats.

SummaryHerpes simplex virus (HSV) mutants kill dividing tumor cells but spare non-proliferating, healthy brain tissue and may be useful in developing new treatment strategies for malignant brain tumors. Two HSV mutants, a thymidine kinase deficient virus (TK-) and a ribonucleotide reductase mutant (RR-), killed 7/7 human tumor cell lines in tissue culture. The TK-HSV killed Rat RG2 glioma and W256 carcinoma lines but not the rat C6 glioma in culture. TK-HSV replication (12 pfu/cell) was similar to wild-type HSV (10 pfu/cell) in rapidly dividing W256 cells in tissue culture, but was minimal (<1 pfu/cell) in serum-starved cells, suggesting that the proliferative activity of tumor cells at the site and time of TK-HSV injection may influence efficacyin vivo. Subcutaneous W256 tumors in male Sprague-Dawley rats were injected with TK-HSV or virus free inoculum. A significant effect of TK-HSV therapy on W256 tumor growth was demonstrated compared to controls (p=0.002). Complete regression was observed in 4/9 experimental tumors, with no recurrence over 6 months. Tumor growth in the remaining 5/9 animals was attenuated during the first 3 to 5 days after treatment, but not beyond 5 days compared to 9 matched control animals; no tumor regression was observed in any of the control animals. These results suggest that HSV mutants are potentially useful as novel therapeutic agents in the treatment of tumors in immunocompetent subjects.

The Velocities of Red Cell and Plasma Flows through Parenchymal Microvessels of Rat Brain are Decreased by Pentobarbital

Local cerebral blood flow is lowered in many brain areas of the rat by high-dose pentobarbital (50 mg/kg). In the present study, the mechanism of this flow change was examined by measuring the distribution of radiolabeled red blood cells (RBCs) and albumin (RISA) in small parenchymal microvessels and calculating the microvascular distribution spaces and mean transit times of RBCs, RISA, and blood. In most brain areas, pentobarbital slightly decreased the RISA space, modestly increased the RBC space, and did not alter the blood space. The mean transit times of RBCs, RISA, and blood through the perfused microvessels were considerably greater in treated rats than in controls. These findings indicate that the mechanism by which high-dose pentobarbital diminishes local cerebral blood flow in rat brain is, in the main, a lowered linear velocity of plasma and RBC flow through small parenchymal microvessels and not decreased percentage of perfused capillaries (capillary retirement). This response is probably driven mainly by lowered local metabolism and may well entail a slight increase in the number of small microvessels that are perfused by RBCs.

Antenatal steroids decrease blood-brain barrier permeability in the ovine fetus

Antenatal corticosteroid therapy reduces the incidence of intraventricular hemorrhage in premature infants. Enhanced microvascular integrity might provide protection against intraventricular hemorrhage. In the adult, there is evidence to suggest that the blood-brain barrier may be under hormonal control. We hypothesized that antenatal corticosteroids decrease blood-brain barrier permeability in the preterm ovine fetus. Chronically instrumented 120-day-gestation fetuses were studied 12 h after the last of four 6-mg dexamethasone (n = 5) or placebo (n = 6) injections had been given over 48 h to the ewes. Blood-brain barrier function was quantified with the blood-to-brain transfer constant (Ki) for alpha-aminoisobutyric acid (AIB). Ki was significantly lower across brain regions in the fetuses of ewes that received antenatal dexamethasone compared with placebo (ANOVA; interaction, F = 2.54, P < 0.004). In fetuses of dexamethasone- and placebo-treated ewes, Ki (microliter . g brain wt-1. min-1, mean +/- SD) was, respectively, 2.43 +/- 0.27 vs. 3.41 +/- 0.74 in the cortex, 4.46 +/- 0.49 vs. 5.29 +/- 0.85 in the cerebellum, and 3.70 +/- 0.49 vs. 5.11 +/- 0.70 in the medulla. We conclude that antenatal treatment with corticosteroids reduces blood-brain permeability in the ovine fetus.Antenatal corticosteroid therapy reduces the incidence of intraventricular hemorrhage in premature infants. Enhanced microvascular integrity might provide protection against intraventricular hemorrhage. In the adult, there is evidence to suggest that the blood-brain barrier may be under hormonal control. We hypothesized that antenatal corticosteroids decrease blood-brain barrier permeability in the preterm ovine fetus. Chronically instrumented 120-day-gestation fetuses were studied 12 h after the last of four 6-mg dexamethasone ( n = 5) or placebo ( n = 6) injections had been given over 48 h to the ewes. Blood-brain barrier function was quantified with the blood-to-brain transfer constant ( K i) for α-aminoisobutyric acid (AIB). K i was significantly lower across brain regions in the fetuses of ewes that received antenatal dexamethasone compared with placebo (ANOVA; interaction, F = 2.54, P < 0.004). In fetuses of dexamethasone- and placebo-treated ewes, K i(μl ⋅ g brain wt-1 ⋅ min-1, mean ± SD) was, respectively, 2.43 ± 0.27 vs. 3.41 ± 0.74 in the cortex, 4.46 ± 0.49 vs. 5.29 ± 0.85 in the cerebellum, and 3.70 ± 0.49 vs. 5.11 ± 0.70 in the medulla. We conclude that antenatal treatment with corticosteroids reduces blood-brain permeability in the ovine fetus.

Cerebral glucose utilization and blood flow in adult spontaneously hypertensive rats.

Not only blood pressure but also behavioral activity, brain morphology, and cerebral ventricular size differ between young spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats. This suggests that cerebral blood flow and cerebral metabolism may vary between these two rat strains. To test this hypothesis, we measured local cerebral glucose utilization in 31 brain areas of 26-30-week-old rats. Local cerebral blood flow was also assessed in these same areas. Cerebral glucose utilization was measured by the 2-deoxyglucose method; cerebral blood flow was determined by the iodoantipyrene method. In virtually all gray matter structures, the apparent rate of glucose utilization was lower in SHR than in normotensive WKY rats; the interstrain differences varied significantly among structures and were statistically significant (uncorrected t tests) in 14 of 28 gray matter areas. Local cerebral blood flow was fairly similar in the two rat strains. The coupling of blood flow to glucose utilization varied significantly among brain areas in normotensive WKY rats as well as in SHR. In a number of gray matter structures, the coupling of flow to metabolism differed between hypertensive and normotensive animals. These data suggest that for many brain areas, either glucose utilization or glucose partitioning differs between WKY rats and SHR.

A heterogenous, pore-vesicle membrane model for protein transfer from blood to cerebrospinal fluid at the choroid plexus

Abstract A negative relation exists between the steady-state, cerebrospinal fluid/blood concentration ratio and the hydrodynamic radius of blood-derived proteins. This negative relation can be explained if blood proteins enter spinal fluid only at the choroid plexus (and not at other sites within the brain), and if transfer at the plexus is limited by the choroidal epithelium and not by demonstrably leaky capillaries within the choroidal stroma. The most likely interpretation of the data is that there are two pathways for protein transfer at the plexus epithelium, a set of 117-A-radius pores which allow transfer of smaller proteins by diffusion, ultrafiltration, or both mechanisms, and a set of 250-A-radius pinocytotic vesicles which account for exchange of larger proteins as well. On the basis of the data, it is possible to calculate permeability and/or osmotic reflection coefficients associated with porous transfer, as well as a rate of vesicular transport. The latter is estimated to be so low that ultrastructural studies would be expected to demonstrate only occasional vesicular discharge from the choroidal epithelium into cerebrospinal fluid. Pores with a 117-A-radius have not been described at the choroidal epithelium, but may represent a 0.08% defect in the normally continuous tight junctions that surround and closely connect choroidal epithelial cells.