In situ freezing of the brain for metabolic studies: evaluation of the "box" method for large experimental animals

The box method of freezing the brain in situ was assessed in baboons. The cooling rate of the tissue was monitored in several regions located at various depths from the skull surface. These measurements allowed us to examine the time required for the tissue to reach 0 degree C, in relation to its depth measured from the top of the skull. To define brain regions with proven ischaemia, frozen tissue sections were surveyed for areas of decreased pH. In addition, concentrations of ATP, phosphocreatine, and lactate were determined in gray matter located at various depths from the top of the brain surface. Normal tissue pH and low lactate concentration, without any significant decrease in high-energy phosphate levels, were found in regions at a depth less than approximately 10 mm from the brain surface. Deep structures including the inferiomedial aspect of the temporal lobe, the lateral geniculate body, and the limbic system (hippocampus) consistently showed mild tissue acidosis, indicating that these regions were subjected to some degree of ischaemia before they were reached by the freezing front. In some cases, acidosis was also detectable in the thalamus, basal ganglia, and in the deeper part of some sulci. We conclude that, with baboons, in situ freezing using the box method is valid for metabolic studies of the cerebral cortex and structures located at a depth less than approximately 10 mm from the top of the brain surface.