Steven Weber (University of Pittsburgh)

Abstract: Analytical chemistry is fundamental to understanding brain function. Better analytical chemistry leads to better understanding. We have been attempting to improve one very common technique, microdialysis sampling with liquid chromatography (LC) measurements, and to develop another, electro-osmotic push-pull perfusion. In both cases, application of fundamental analytical chemistry principles including mass transport, separations, and electrochemistry are necessary. In the former technique, the microdialysis probe is perfused with artificial cerebrospinal fluid (aCSF). Solutes in the brain diffuse across the membrane and are swept away to an instrument for measurement. The typical time resolution of this technique is 5 – 60 minutes, with 15 – 20 heavily favored. To achieve better time resolution and maintain the required sensitivity requires adapting the LC to smaller samples (because the aCSF flow rate is more or less fixed) while maintaining a fixed number of theoretical plates. Application of theory prescribes capillary columns in the 100 m diameter range 3 – 4 cm long using the smallest available packing particles and elevated temperature. Optimizing the electrochemical detector potential aids sensitivity and reproducibility.In the latter technique, we seek to determine rates of extracellular processes by passing a solute through the tissue and determining quantitatively how much is altered (enzymatic degradation) or missing (uptake into cells). Based on theory and our determination that brain tissue has a significant zeta potential, using the electrochemical technique of electro-osmosis should be a good way to reproducibly perfuse tissue in a small region. We have applied this technique to the determination of the rate of hydrolysis of the neuroprotective peptide leucine enkaphalin. We demonstrated that two regions of the hippocampus that differ in their susceptibility to stroke (lack of blood flow) also differ in the enzyme activity of an ectopeptidase that cleaves leucine encephalin which could explain the observed differences in their susceptibility.

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