FREEZE‐BLOWING: A NEW TECHNIQUE FOR THE STUDY OF BRAIN <i>IN VIVO</i>

Abstract

Abstract— A new apparatus is described which removes and freezes brains of conscious rats more rapidly than was heretofore possible. The apparatus consists of two probes which are driven simultaneously into the cranial vault of the rat immobilized in a specially constructed restraining cage. When in position, air under pressure enters through one probe and blows the supratentorial portion of the brain tissue (situated between the olfactory bulbs and the superior colliculi) out the other probe and into a thin chamber previously cooled in liquid N 2 . This method stops brain tissue metabolism more rapidly than the previously‐described methods of microwave irradiation, decapitation into liquid N 2 , or whole‐animal immersion into liquid N 2 , as evidenced by the measurement of labile metabolites and redox states. Thus, samples of freeze‐blown brain had higher levels of a‐oxoglutarate, creatine phosphate, pyruvate, glucose and glucose‐6‐phosphate and lower levels of lactate, malate and AMP than brain tissue obtained by the other methods. The free cytoplasmic [NAD + ]/[NADH 2 ], [NADP + ]/[NADPH 2 ] and [ATP]/[ADP] [HPO 4 2‐ ] ratios were higher in freeze‐blown samples. These data indicate that more extensive anoxic metabolism occurred when methods other than freeze‐blowing were used. We conclude that the levels of metabolites measured in brain obtained with the freeze‐blowing technique more closely resemble those which occur in vivo.