The widely accepted concept of the ‘protective cutting' method:
benefits and present limitations
For several decades the vast majority of brain slice physiologists have relied upon a “protective cutting method” for preparing healthy brain slices from juvenile and adolescent animals. This method is based on the premise that passive Na influx and subsequent water entry and cell swelling during the slice cutting step is the major insult that leads to poor survival of neurons, particularly for those neurons located in the superficial layers that are most likely to sustain direct trauma from the blade movement. Thus the implementation of a protective cutting solution having equa-molar replacement of NaCl with sucrose (e.g. low Na+ aCSF, sucrose-substituted aCSF, or simply sucrose aCSF) provides a notable improvement in neuronal preservation, especially for difficult to preserve brain regions such as brainstem and other highly myelinated regions [Aghajanian and Rasmussen, 1989 Synapse].
The sucrose aCSF protective cutting solution has been widely adopted for preparing acute brain slices from virtually all brain regions following the initial description over twenty years ago. Variations of the protective cutting method have subsequently been described, including modified protective cutting aCSF formulations with mixed NaCl/sucrose or implementing alternative Na-substitutes such as choline, NMDG, Tris, or glycerol. Different studies have reported improved neuronal viability in acute brain slices using these different protective cutting solutions, but no clear consensus has emerged in the field to support a most effective formulation. In our recent work we have had reasonable success in using all of these various protective cutting solutions to prepare acute brain slices from juvenile mice. However, when we attempted to move into acute slice preparation from mature adult animals we experienced that none of the existing methods or modified aCSF formulations provided an acceptable quality of brain slice for routine electrophysiological analysis.