Use of ascorbate in the preparation and maintenance of brain slices

Ascorbate and glutathione (GSH) are normally concentrated in brain cells at millimolar levels. However, both of these low-molecular-weight antioxidants are washed out of mammalian brain tissue during slice preparation and subsequent incubation. Ascorbate, which is not synthesized in the brain, can be added back to slices by active uptake from the incubation medium. Levels of GSH, on the other hand, are regulated by synthesis rather than uptake, and cannot be readily maintained in slices. Importantly, maintenance of brain slice ascorbate content at at least 50% of that in vivo, prevents the increase in slice water content that normally occurs during incubation. Slices with maintained ascorbate levels also have better histological characteristics than ascorbate-depleted tissue. The medium concentration of ascorbate sufficient to maintain content and inhibit edema formation is 400 microM, which is the normal concentration in brain extracellular fluid. This paper describes methods to maintain ascorbate levels in brain slices, including procedures to minimize oxidation in oxygenated incubation media. Also described is an HPLC analysis for ascorbate and GSH that is based on direct injection rather than extraction of samples.