METABOLIC CHANGES IN THE BRAINS OF MICE FROZEN IN LIQUID NITROGEN

Abstract— Autolytic changes in the mouse brain, occurring during immersion of the animal in liquid nitrogen, were evaluated by measuring the tissue concentrations of glucose, lactate, pyruvate, α-oxoglutarate, phosphocreatine, creatine, ATP, ADP and AMP. The values thus obtained were compared with those obtained in paralysed mice under nitrous oxide anaesthesia, the brains of which were frozen in such a way that arterial blood pressure and oxygénation were upheld during the freezing. Immersion of unanaesthetized mice in liquid nitrogen gave rise to significant alterations in phosphocreatine, creatine, lactate, lactate/pyruvate ratio, ADP and AMP. A comparison with values obtained in paralysed and anaesthetized mice that were frozen by immersion in liquid nitrogen showed that the metabolic changes observed in the unanaesthetized animals could not be caused by an anaesthetic effect on the metabolic pattern. It is concluded that autolysis in the mouse brain occurs during immersion of the animal in a coolant, mainly because arterial hypoxia develops before the tissue is frozen. A comparison with previous results on rat cerebral cortex indicates that mice offer no advantage for studies of cerebral metabolites in unanaesthetized animals. In both species, accurate analyses of labile cerebral metabolites require that the brain is frozen in a way that prevents arterial hypoxia during the fixation of the tissue.     

Biochemical and genetic evidence for a macromolecular -glucuronidase complex in microsomal membranes

In the mouse β-glucuronidase is present in both microsomes and lysosomes and the enzyme at both sites is coded by the same structural gene. Electrophoresis on polyacrylamide gels showed that liver, kidney and lung from normal strains contained five enzyme forms designated L, M1, M2, M3 and M4 in order of decreasing mobility toward the anode. Band L is found primarily in lysosomes and is a tetramer of 260,000 molecular weight. Bands M1 to M4 are found exclusively in microsomes and range in molecular weight from 310,000 to 470,000. The increase in molecular weight is due to sequential addition of an accessory protein chain. When glucuronidase is highly induced in kidneys of female mice by injection of dihydrotestosterone, a sixth electrophoretic form of glucuronidase, designated X, appears. Form X appears early in induction, is localized in microsomes, and has a molecular weight (260,000) equal to that of the tetramer form L.Mice homozygous for the eg ° mutation, and thus deficient in microsomal glucuronidase, completely lack the microsomal forms M1 to M4. They do contain form X, and this increases after testosterone induction in kidney. The form X present in eg ° mice is indistinguishable from the form X seen in normal induced kidney.It appears that mice synthesize two different tetrameric forms of glucuronidase from the same structural gene. One, form L, is lysosomal; the other, form X, gives rise to microsomal enzyme forms M1 to M4 by the successive addition of up to four accessory protein chains. The eg ° mutant is blocked in the conversion of X to M1.