Brain slice viability determined under normoxic and oxidative stress conditions: involvement of slice quantity in the medium

Objective: In vitro acute adult brain slice methods are instruments in developing our knowledge of the nervous system. Optimization of this method for obtaining high-quality brain slices is extremely important in terms of consistency and reliability of the experimental results. Although some important topics such as slice thickness, temperature, and composition of the physiological medium have been studied for optimization, involvement of slice quantity in medium on tissue viability has not been investigated yet. Methods: Different number of slices (1, 3, or 6 slices) were incubated under normoxic or some prooxidant stress conditions induced by oxygen–glucose deprivation (OGD), H₂O₂, FeSO₄+ ascorbic acid, or menadione to evaluate the effect of slice density on tissue viability. Results:Slice quantity in the normoxic incubation medium caused a significant increase in 2,3,5-triphenyltetrazolium chloride (TTC) staining intensity of the slices. Similarly, increase in the slice quantity in the medium also protected the slices against either OGD, H₂O₂, FeSO_4, or menadione-induced decrease in TTC staining. In addition to TTC staining, lactate dehydrogenase leakage or malondialdehyde and reactive oxygen species production under normoxic or ischemia-like conditions were also attenuated by increasing slice quantity in the medium. Conclusion: These results show that when using brain slices method for investigating the structural and functional features of brain at the molecular and cellular levels, both slice quantity in the medium and incubation volume should be considered first. Increasing slice quantity or decreasing incubation volume probably causes an increase in the concentration of endogenous substance(s) involved in neuroprotection.