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 antioxidant s are washed out of mammalian brain tissue during slice preparation and sub sequent incubation. ascorbate, which is not synthesized in the brain, can b e 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, a nd cannot be readily maintained in slices. Importantly, maintenance of brai n slice ascorbate content at at least 50% of that in vivo, prevents the inc rease in slice water content that normally occurs during incubation. Slices with maintained ascorbate levels also have better histological characteris tics than ascorbate-depleted tissue. The medium concentration of ascorbate sufficient to maintain content and inhibit edema formation is 400 mu M, whi ch is the normal concentration in brain extracellular fluid. This paper des cribes methods to maintain ascorbate levels in brain slices, including proc edures to minimize oxidation in oxygenated incubation media. Also described is an HPLC analysis for ascorbate and GSH that is based on direct injectio n rather than extraction of samples. (C) 1999 Academic Press.