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.

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.