Optimizing dynamic T2* MR imaging for measurement of cerebral blood flow using infusions for cerebral blood volume.

We describe an approach to measuring cerebral blood flow (CBF) based on independent measurements of cerebral blood volume (CBV) and mean transit time (MTT) with calculation of CBF by using the central volume theorem: CBF = CBV / MTT. This permits optimization of the individual acquisitions and analyses. In particular, measurement of CBV during contrast infusion, rather than simultaneously with MTT from a single bolus, yields values more consistent with those of other methods.

Thick brain slices model the ischemic penumbra.

Hypothalamic brain slices, varying in thickness from 400 mu to 1,000 mu, were assessed by studying 2-deoxyglucose (2DG) metabolism, lactate accumulation, inulin spaces, and morphology at the light and ultrastructural levels. Evidence of increased glycolytic flux due to anaerobic metabolism is found at thickness greater than 600 mu in association with a progressive increase in the inulin-exclusion space. The metabolic profiles, as a function of depth into the slices, reveal that 700-mu slices function in a manner similar to 540-mu slices at the surfaces, but with a core of increased 2DG phosphorylation at the slice center. In contrast, the 1,000-mu slices show significant reduction of 2DG and increases in 2DG6P relative to the 540-mu slices at the slice surface as well as in the slice interior, suggesting impaired transport of 2DG into cells and spread of ischemic injury from the slice interior to the slice surface. Despite these metabolic changes, only minor morphologic changes of ischemic injury were found at the center of thicker slices, and in vitro glucose utilization of 1000-mu slices remained constant for up to 15 h. These three slice thicknesses should provide a useful model for studying the neurochemistry and neuropharmacology of the ischemic penumbra.

Ischemic brain slice glucose utilization: effects of slice thickness, acidosis, and K+.

Brain slices of varying thickness were used to modify retention of metabolic products in an in vitro model of ischemia. Past and present results reveal increased anaerobic glycolysis in 660-microns slices with accumulation of lactate as slice thickness reaches 1,000 microns. Brain slice glucose utilization and lactate content were measured in buffers of various extracellular K+ levels and pH in 540-, 660-, and 1,000-microns slices. Acidosis suppresses glucose utilization at all slice thicknesses without affecting tissue lactate. Studies of 2-deoxyglucose metabolites establish that the suppression of glucose utilization by acidosis is due entirely to inhibition of glucose phosphorylation without any effect on glucose uptake into tissue. The inhibition is reversible after 45 min at pH 6.1. The experiments with acidosis also suggest that persistent energy demands continue to stimulate phosphofructokinase despite the low pH so that glycolysis continues, with potential for injury. Increasing K+ increases glucose utilization and tissue lactate at all three thicknesses. Correlations of glucose utilization with lactate accumulation support the possibility that high K+ may exert a dual influence on the tissue metabolism, not only stimulating glucose utilization by inducing depolarization but also by influencing the removal of metabolic products.

Kinetic model of 2-deoxyglucose metabolism using brain slices.

A six-compartment, nine-parameter kinetic model of 2-deoxyglucose (2DG) metabolism, which includes bidirectional tissue transport, phosphorylation, two-step dephosphorylation, phosphoisomerization, and conjugation to UDP and macromolecules, has been derived. Data for analysis were obtained from 540- and 1,000-microns-thick hippocampal and hypothalamic brain slices, which were incubated in buffer containing [14C]2DG, frozen, extracted with perchlorate, and separated on anion-exchange columns. Solutions of the equations of the model were fit to the data by means of nonlinear least-squares analysis. These studies suggest that dephosphorylation is adequately described by a single reaction so that the model reduces to eight parameters. The in vitro rate constants for transport, phosphorylation, and dephosphorylation are very similar to prior in vivo results. The phosphoisomerization rate constant is similar to dephosphorylation, so glycosylated macromolecules slowly accumulate and gradually assume larger relative importance as other compounds disappear more rapidly. Rate constants for 540-microns slices from hypothalamus and hippocampus are similar, while 1,000-microns slices have smaller tissue transport constants and larger phosphorylation constants. The rate equation for glucose utilization of this model is relatively insensitive to uncertainties regarding the rate constants. Including later metabolic components in kinetic models improves the calculations of glucose utilization with long isotope exposures.

Simplified brain slice glucose utilization.

Brain slice glucose utilization (SGU) can be measured by methods analogous to those used for in vivo cerebral glucose utilization. In order to make this technique more accessible and applicable to a broad range of experimental conditions, we have derived a simplified operational rate equation and generated the table of apparent rate coefficients necessary to apply the equation under different experimental situations. Calculations of the apparent rate coefficients were based upon an eight-parameter kinetic model combined with Michaelis-Menten theory to account for changes in the rate constants as a function of buffer glucose concentration. The theory was tested with a series of experiments using rat brain slices. [14C]-2-deoxyglucose (2DG) and [14C]-3-O-methylglucose (3OMG). The errors involved in the simplified technique were estimated by a variety of techniques and found to be acceptable over a broad range of conditions. A detailed, practical protocol for the simplified method is presented.

Diffusion of radiotracers in normal and ischemic brain slices.

Diffusion in the extracellular space (ECS) is important in physiologic and pathologic brain processes but remains poorly understood. To learn more about factors influencing tissue diffusion and the role of diffusion in solute-tissue interactions, particularly during cerebral ischemia, we have studied the kinetics of several radiotracers in control and hypoxic 450-microm hippocampal slices and in 1,050-microm thick slices that model the ischemic penumbra. Kinetics were analyzed by nonlinear least squares methods using models that combine extracellular diffusion with tissue compartments in series or in parallel. Studies with 14C-polyethylene glycol confirmed prior measurements of extracellular volume and that ECS shrinks during ischemia. Separating diffusion from transport also revealed large amounts of 45Ca that bind to or enter brain as well as demonstrating a small, irreversibly bound compartment during ischemia. The rapidity of 3H2O entry into cells made it impossible for us to distinguish intracellular from extracellular diffusion. The diffusion-compartment analysis of 3-O-methylglucose data appears to indicate that 5 mmol/L glucose is inadequate to support glycolysis fully in thick slices. Unexpectedly, the diffusion coefficient for all four tracers rose in thick slices compared with thin slices, suggesting that ECS becomes less tortuous in the penumbra.

Restoring adenine nucleotides in a brain slice model of cerebral reperfusion.

Tissue adenine nucleotides are depleted during cerebral ischemia, impeding recovery after reperfusion. Although prior studies have attempted to prevent the initial loss of adenylates, the present study tests the hypothesis that stimulating synthesis of adenine nucleotides, through either adenosine kinase or adenine phosphoribosyltransferase, would result in significant cerebroprotection. To study the effects on neurons and glia directly while avoiding the influence of the cerebral vasculature, hippocampal brain slices were used for the model of transient ischemia with reperfusion. The standard brain slice insult of brief exposure to anoxia with aglycemia was modified based on studies which showed that a 30-minute exposure to air with 1 mmol/L glucose produced a stable, moderate reduction in ATP during the insult and that, 2 hours after return to normal conditions, there was moderate depletion of tissue adenine nucleotides and histologic injury. Treatments with 1 mmol/L adenosine, AMP, or adenine were equivalent in partially restoring adenine nucleotides. Despite this, only adenosine afforded histologic protection, suggesting a protective role for adenosine receptors. There also was evidence for metabolic cycling among adenine nucleotides, nucleosides, and purines. Adenosine may exert direct cerebroprotective effects on neural tissue as well as indirect effects through the cerebral vasculature.

Glucose utilization of ischemic hippocampal slices.

Hippocampal brain slices that were 1000 mu thick were prepared from Sprague-Dawley rats and studied using in vitro glucose utilization under well-oxygenated conditions or after a 15 min anoxic insult produced with a nitrogen atmosphere. Autoradiography reveals that glucose utilization is increased in CA1 and CA3 stratum radiatum of 1000 mu slices, even with full oxygenation, compared to the same regions in 540 mu slices. Following anoxia, there is an initial addition increase in stratum oriens of CA1 and CA3 glucose utilization that is followed by a decline in glucose utilization in all slice regions within an hour of the insult. Because increased glucose utilization is apparent at the slice surfaces as well as at the interior, it is suggested that thick brain slices are a model of brain ischemia, not just hypoxia.

Effects of dextran on hippocampal brain slice water, extracellular space, calcium kinetics and histology.

Hippocampal brain slices are valuable models for studying brain function but are compromised by several artifacts, including significant water gain and histologic injury, which occur under certain incubation conditions. Addition of colloid to Krebs-Ringer buffer (K-R) has been shown to eliminate water gain but has not achieved widespread acceptance. We confirm prior observations that dextran and PEG lessen the increase in slice mass during incubation in a dose-dependent manner with no water gain occurring at 4% concentrations. However, we also observe that addition of colloid to standard K-R induces severe neuronal pyknosis. Fortunately, the pyknosis can be eliminated by reduction in buffer osmolarity through adjustment of NaCl, producing markedly improved slice histology in dextran buffer, especially in the CA3 and CA4 regions of the hippocampus which are severely injured when incubated submerged in K-R at 37 degrees C. Extracellular space markers are not affected by either colloid. The volume of distribution for 45Ca is much larger in dextran buffers than in K-R and variability of 45Ca kinetics is also reduced. In the presence of dextran, hypoxia induces significant slice water gain, a relatively selective histologic injury and an alteration of tissue Ca2+ kinetics. Use of dextran buffers may eliminate many troubling brain slice artifacts.

Effects of K+, pH and glutamate on 45Ca kinetics in hippocampal brain slices.

Altered calcium homeostasis is likely to play a pathogenetic role in cerebral ischemia. In order to further understand which factors associated with ischemia contribute to disturbances of calcium metabolism, the influence of 3 isolated insults, 8 mM K+, pH 6.1 and 1 mM glutamate, on total tissue calcium were studied by analysis of steady-state kinetics of 45Ca in 500 microns hippocampal brain slices. 45Ca kinetics were analyzed with 2 bi-exponential models by non-linear least-squares analysis. Tissue wet weight/protein was measured simultaneously. Each experimental condition produced a unique tissue response. Raising K+ had no effect on tissue water but increased the rate of uptake of Ca2+ into the larger, rapidly equilibrating tissue Ca2+ space. Acidosis reduced tissue water and the amount of Ca2+ in the slowly equilibrating compartment due to enhanced efflux from that space. Glutamate increased tissue water in a time-dependent manner and increased the influx and amount of Ca2+ in the slowly equilibrating space. Combined insults revealed minimal interaction between K+ and acidosis or glutamate, but glutamate with acidosis worsened tissue injury. We discuss the relationship of this technique to other methods for studying tissue calcium and the significance of the observations regarding ischemia.

Rhythm of suprachiasmatic nucleus 2-deoxyglucose uptake in vitro.

Using a hypothalamic slice preparation containing the suprachiasmatic nucleus (SCN) and measurement of 2-deoxy[14C]glucose (2-DG) uptake by autoradiography, we have demonstrated that, after 1 h in vitro, 2-DG uptake into the SCN is proportional to the rate of glucose utilization present in vivo at the corresponding subjective time of day and that, during an 8-h incubation in vitro, the SCN is capable of spontaneously changing its metabolic rate.

Preservation of hippocampal brain slices with in vivo or in vitro hypothermia.

Hippocampal brain slices show CA1 injury similar to that seen after global ischemia in vivo. Cooling rats to 31 degrees C prior to sacrifice or cooling slices to 21 degrees C for 45 min increased the percentage of normal CA1 pyramidal cells after 5 h in vitro from 30% to over 80%. Brain slices also show a unique, consistent injury in dentate which is lessened by transient cooling to 21 degrees C but not by cooling the animal.

Two augmentative communication systems for speechless disabled patients.

Patients may be rendered speechless because of many conditions, including cancer surgery, stroke, cerebral palsy, cervical cord and head trauma, neuromuscular paralysis, and intubation for respiratory failure. These same conditions may also be associated with decreased use of the hands, so that writing and other nonverbal forms of communication are also impaired. Lack of communication can frustrate the patient, the family, and health care personnel; increase the patient's isolation; and lead to poor patient cooperation, thus impeding progress in therapy and producing secondary psychiatric disturbances. Two communication programs that use a Commodore 64 computer are described in this paper. One communication program uses the alphabet and the other is based on the international Morse code. These programs are easy to use and inexpensive to establish, and they accommodate any switching device.

Brain slice glucose utilization.

The metabolism of 2-deoxyglucose has been studied in 540 micron and 1,000 micron hypothalamic brain slices. Slice 2-deoxyglucose (2DG) and 2-deoxyglucose-6-phosphate (2DG6P) levels were measured after tissue homogenization and perchloric acid extraction. By analyzing the uptake and washout kinetics with nonlinear least-squares methods, we have determined the rate constants for three-, four-, or five-parameter kinetic models and obtained a value for the in vitro lumped constant (LC). The kinetic analysis reveals a small, slowly decaying, 2DG component that is not predicted by any of the models. If this component is treated as a separate, parallel compartment, then the four- and five-parameter models are essentially equivalent. To compare our data to prior in vivo data, we combined 2DG and 2DG6P to produce Ci*, the total slice radioactivity, and analyzed the first 45 min of uptake. These data were fit best by a three-parameter model and the slowly decaying pool was not identified. Calculation of glucose utilization from total tissue radioactivity, measured by whole slice homogenization and by image analysis of autoradiograms, showed excellent correlation between the two methods. Image analysis of radioactivity in the suprachiasmatic nucleus, which is present in these slices, revealed a spontaneous diurnal variation in in vitro glucose utilization in close quantitative agreement with prior in vivo measurements. The kinetic analysis of the 1,000 micron slice was qualitatively similar to that of the 540 micron slice but revealed an increase in the LC and a large decrease in k1 as well as the expected large increase in the hexokinase rate constant, k3. Overall, in vitro glucose utilization increased by about 60%. These results are consistent with our prior studies of the 1,000 micron slice and support our interpretation that the 1,000 micron slice is an excellent in vitro model for brain ischemia without infarction.

Analysis of in vitro glucose utilization in a circadian pacemaker model.

An in vitro glucose utilization method, based upon 14C-2-deoxyglucose kinetics in brain slices, has been used to study circadian rhythms in hypothalamic slices containing the suprachiasmatic nucleus (SCN). Spontaneous SCN metabolic activity in vitro is similar to that observed in vivo with higher metabolic rates in subjective daytime and lower rates during subjective night. However, in vitro SCN metabolic activity during late subjective day is above that seen when glucose utilization is measured in vivo, suggesting that an inhibitory influence normally active in vivo is lost during slice isolation. Incubation of slices containing SCN in the presence of TTX exposes a TTX-insensitive component of metabolic activity in early subjective day, supporting prior suggestions that glucose utilization by the circadian oscillator continues in the absence of Na(+)-dependent action potentials. Studies with high Mg2+ concentrations are consistent with the hypothesis that most metabolic activity above the basal level observed with the glucose utilization method is related to synaptic activity. Pharmacological studies of the SCN brain slice model with radiotracers offer potential for analysis of both circadian rhythmicity and neural regulation.

Cerebral blood volume measurements by T*2-weighted MRI and contrast infusion.

A reliable, accurate, and accessible method for measuring cerebral blood volume (CBV) has been developed based on T(*) (2)-weighted MRI and a 1-min infusion of gadolinium instead of a bolus. Computer simulations predict that this infusion CBV method will have a signal-to-noise ratio (SNR) 3-5 times greater than that obtained by area-under-the-curve (AUC) methods, with high accuracy over a wide range of arterial, tissue, and MRI conditions. In six healthy controls, the CBV was 1.87 +/- 0.44 in white matter (WM), 3.40 +/- 0.44 in deep gray matter (DGM), and 3.84 +/- 1.87 ml blood/100 g tissue in cortical GM (CGM). The mean GM/WM ratio was 1.94. In five patients with bilateral carotid disease, the corresponding values were 2.63 +/- 0.33, 4.72 +/- 0.33, and 5.27 +/- 2.40 ml blood/100 g tissue, all of which were significantly different from controls. AUC values were generally higher and failed to demonstrate differences between controls and patients. The infusion method shows great potential for providing reliable, accurate, and accessible CBV values with the ability to discriminate physiologic or pathological volume changes under a wide range of conditions.