Modifications for aging animals and difficult brain regions
In order to counteract the glutathione depletion for better functional and morphological preservation of brain slices from aging animals, the NAC or GSH-EE can be added directly into the modified aCSF throughout the stages of slice incubation. We have investigated addition of NAC (12 mM) or GSH-EE (3 mM) at various stages to try to determine the critical step where the majority of the benefit is derived. So far it seems that inclusion of NAC or GSH-EE at any stage alone can have a notable benefit, with no particular stage being exceptionally more important than the rest. Our feeling is that it is important to get the glutathione levels restored as early as possible to avoid irreversible damage (such as lipid peroxidation), so the earlier the exposure to NAC or GSH-EE the better. This is why we advocate for inclusion of NAC or GSH-EE in the perfusion, slicing, initial recovery, and long-term holding chamber. The NAC and GSH-EE are never included in the recording aCSF.
Please also note that the choice of modified aCSF (NMDG-HEPES or choline-HEPES) depends on the exact age and brain region under investigation. All formulations, including traditional sucrose aCSF formulations, are compatible with the strategies for glutathione restoration we have described. Notably, we find a synergistic effect of combining the protective recovery method and glutathione restoration strategy--leading to the healthiest adult brain slices of any method that has been described to date. We hypothesize that the improved neuronal preservation with the protective recovery method may lead to higher synaptic activity (more functional synapses preserved) and thus predispose slices to higher risk of damage from excitotoxicity over time. Therefore, the glutathione restoration strategy is highly effective when used in combination with the protective recovery method by counteracting oxidative stress and allowing neurons to resist damage over a greatly extended timeframe.
Other tips and notes: We have used high concentrations of NAC and GSH-EE in our experiments to ensure maximal neuronal glutathione restoration at early time points. Lower concentrations will likely be effective or sufficient (e.g. NAC 1-5 mM or GSH-EE 0.5-1 mM), particularly for brain slice preparation from young adult animals as opposed to aged animals. It is critical to ensure that the pH and osmolarity are maintained in the correct range after addition of these supplements. 12 mM NAC will drop the pH to 6.9 and increase osmolarity to 320 mOsm. We adjust pH with NMDG powder and reduce osmolarity with a small amount of pure water. GSH-EE will not significantly alter pH or osmolarity at the recommended concentrations.