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.     

CEREBRAL ENERGY STATE IN INSULIN-INDUCED HYPOGLYCEMIA, RELATED TO BLOOD GLUCOSE AND TO EEG

—Concentrations of phosphocreatine, creatine, ATP, ADP and AMP were measured in the cerebral cortex of rats during insulin-induced hypoglycemia. Blood glucose concentrations were related to clinical symptoms in unanaesthetized animals and to the EEG pattern in paralysed and lightly anaesthetized animals. There was an excellent correlation between blood glucose concentration and EEG pattern. In animals showing a pronounced slowing of the EEG or convulsive polyspike activity for up to 20 min, there were no changes in any of the phosphates. However, after prolonged convulsive activity some animals showed clear signs of energy failure, and in all animals with an isoelectric EEG there was a major derangement of the energy state. Since the majority of those animals did not show signs of cerebral hypoxia or ischemia it is concluded that hypoglycemic coma is accompanied by substrate deficiency of a degree sufficient to induce energy depletion of brain tissue.     

INFLUENCE OF ANAESTHETICS ON THE BALANCE BETWEEN PRODUCTION AND UTILIZATION OF ENERGY IN THE BRAIN

Abstract— The influence of general anaesthesia upon the metabolic state of the brain was evaluated from the tissue concentrations of ATP, ADP and AMP, and from the concentrations of glycolytic and citric acid cycle intermediates, in immobilized and artificially ventilated rats anaesthetized either with 70% N₂O, 1% halothane or 60 mg/kg of pentobarbitone. The results were compared to the results obtained on awake animals in fentanyl-analgesia. The adenylate energy charge was identical in all groups studied and there were no H⁺-independent changes in the phosphocreatine/creatine ratios. In pentobarbitone anaesthesia there was an accumulation of glucose 6-phosphate and a fall in fructose 1,6-diphosphate, indicating inhibition of phosphofructokinase. No significant changes in these metabolites were observed with halothane or nitrous oxide anaesthesia and the substrate patterns differed from that obtained with pentobarbitone.
The blood glucose concentrations were higher in the unanaesthetized, immobilized rats given fentanyl than in those anaesthetized. There was a direct relationship between the glucose concentrations in blood and in tissue. The glucose concentration ratios intracellular water to blood were higher in the anaesthetized than in the unanaesthetized animals, increasing with increasing depth of anaesthesia. The intracellular lactate concentrations were lowest in the groups given pentobarbitone and fentanyl citrate, and there was thus no direct relationship between lactate concentration and depth of anaesthesia.     

EVALUATION OF IN SITU FREEZING OF CAT BRAIN BY NADH FLUORESCENCE

Abstract— Cat brain was frozen in situ with liquid nitrogen. In order to locate areas with ischemic artifact, frozen brain slices were surveyed for regions of increased NADH fluorescence. In addition, levels of ATP, phosphocreatine, lactate, and NADH were determined in various brain regions. High levels of ATP and phosphocreatine, and low levels of lactate and NADH were present in all brain regions except the depths of some cortical sulci. These regions of ischemic change were easily detected by virtue of increased NADH fluorescence in frozen brain slices. Deep brain structures such as basal ganglia and hippocampus showed neither high tissue fluorescence nor ischemic changes of the metabolites measured. Therefore, in situ freezing of cat brain adequately preserves metabolite levels in most regions.     

An Improved Method for In Situ Freezing of Cat Brain for Metabolic Studies

This study introduces a new method for rapid freezing of the cat brain. The method employed a Styrofoam box which was fitted around the head of the animal. Liquid nitrogen was poured into the box until the head was submerged. Temperature changes in three brain sites (ventral hypothalamus, the fourth ventricle, and the corpus callosum) and levels of labile carbohydrate metabolites (glycogen, glucose, ATP, P-creatine, and lactate) in five brain regions (cortex, thalamus, midbrain, cerebellum, and pons) frozen by the box method were compared with those frozen by a conventional cup method in which liquid nitrogen was poured into a hollow Styrofoam cup placed on top of the skull. The box method shortened the time of arrival of the freezing front and improved the freezing rate. The time required to bring the tissue to -20 degrees C was shortened, from 20 min at the ventral hypothalamus and 10-12 min at the fourth ventricle with the cup method, to less than 5 min at both sites with the box technique. Continued perfusion of brainstem prior to freezing was demonstrated. Levels of metabolites frozen by either method were similar. Lactate levels in any of the five brain regions studied by either method were not elevated, indicating no ischemic change.(ABSTRACT TRUNCATED AT 250 WORDS)     

Persistence of central respiratory rhythmogenesis after maximal acetylcholinesterase inhibition in unanaesthetized cats

Cats were given systemically the anticholinesterase paraoxon at a dosage (3 mg/kg i.v.) that produced a maximal (over 90%) inhibition of brainstem acetylcholinesterase. All paralyzed and artificially ventilated animals were either unanaesthetized (decerebrated or ventilated with 70% nitrous oxide and 30% oxygen) or anaesthetized (with pentobarbital, alpha-chloralose, or halothane). In unanaesthetized cats, paraoxon produced an immediate rise in arterial blood pressure and did not suppress phrenic nerve respiratory discharges, while in anaesthetized animals it produced an immediate and long-lasting hypotension and a complete arrest of central respiratory activity. It is concluded that acetylcholine accumulation may not suppress respiratory rhythmogenesis and that most anaesthetics may considerably alter the response of cardiorespiratory cholinergic mechanisms to anticholinesterase administration.     

Effects of decapitation, ether and pentobarbital on guanosine 3′,5′-phosphate and adenosine 3′,5′-phosphate levels in rat tissues

Cyclic GMP and cyclic AMP levels in eight different rat tissues were examined after animlas were immersed in liquid nitrogen. In order of decreasing concentration, cerebellu, kidney, lung and cerebral cortex contained the greatest quantities fo cyclic GMP. These tissues also contained relatively high concentrations of cyclic AMP. Compared to values in animals which were sacrificed in liquid nitrogen, levels of both nucleotides in many of the tissues examined were altered by decapitation or anesthesia with ether and pentobarbital. Decapitation increased the levels of both cyclic GMP and cyclic AMP in cerebellum, lung, heart, liver and skeletabl muscle. However, decapitation increased only cyclic AMP in cerebral cortex and kidney. Our previously reported high level of cyclic GMP in lung was attributed to ether anesthesia and surgical removal which increased the cyclic GMP content in lung, heart, testis and skeletal muscle. The effect of ether on cyclic GMP levels in lung and heart was blocked by pretreatment of animals with atropine which indicated that cholinergic agents increase cyclic GMP content in these tissues. Acetylcholine and carbachol in the presence of theophylline increased the accumulation of cyclic GMP in incubations of rat lung minces. Increases in cyclic GMP and cyclic AMP levels in cerebellum with ether anesthesia were prevented if rats were immersed in liquid nitrogen after anesthesis with ether. Anesthesia with pentobarbital decreased the levels of cyclic GMP in cerebellum and kidney and increased the nucleotide in heart, liver, testis and skeletal muscle compared to levels in tissues from animals immersed in liquid nitrogen. However, pentobarbital increased cyclic AMP levels in cerebellum and cerebral cortex and decreased the nucleotide in liver, kidney, testis and skeletal muscle. These studies provide a possible explanation for the variability in in vivo levels of cyclic GMP and cyclic AMP which have been previously reported. In addition, these studies support the hypothesis that the synthesis and degradation of cyclic AMP and cyclic GMP are regulated independently and not necessarily in a parallel or reciprocal manner. These studies also suggest that the increase accumulation of one cyclic nucleotide has no major effect on the synthesis and/or metabolism of the other; however, such interactions cannot be entirely excluded from the results of this study.     

Mitochondrial and cytosolic ATP/ADP ratios in rat liver in vivo.

The ratio of ATP content/ADP content in livers from unanaesthetized fed rat was 0.9 in the mitochondrial matrix and 6.9 in the cytosol; the values for starved (48 h) animals were 1.0 and 5.9 respectively. The mitochondrial ratios observed in unanaesthetized animals were higher than in haemoglobin-free-perfused liver and lower than in isolated hepatocytes. Possible reasons for these differences may be related to oxygen supply and/or other factors. Further, data from anaesthetized rats with the liver exposed are given: mitochondrial ATP/ADP ratios were decreased with pentobarbital, but less so with ketamine as narcotic agent.     

EFFECT OF HYPOTHERMIA UPON ORGANIC PHOSPHATES, GLYCOLYTIC METABOLITES, CITRIC ACID CYCLE INTERMEDIATES AND ASSOCIATED AMINO ACIDS IN RAT CEREBRAL CORTEX

—The influence of hypothermia upon the metabolism of the brain was studied by reducing body temperature in N₂O-anaesthetized rats to 32, 27 or 22°C, with subsequent measurements of organic phosphates, glycolytic metabolites, citric acid cycle intermediates and associated amino acids. Hypothermia was maintained for either 1 or 2 h and the effect of anaesthesia was evaluated by maintaining unanaesthetized animals at 22°C. Hypothermia had no influence on the cerebral cortical concentrations of ATP, ADP or AMP and there was only a small increase in phosphocreatine. Since the tissue concentrations of glucose and glycogen were reduced, it is concluded that the well known resistance of the hypothermie brain to ischaemia is unrelated to increased energy stores. Hypothermia was accompanied by decreases in the tissue concentrations of fructose-1,6-diphosphate, dihydroxyacetone phosphate, 3-phosphoglycerate, pyruvate, lactate, α-ketoglutarate, succinate and malate, but not of glucose-6-phosphate or citrate. These results indicate that metabolic flux is retarded mainly at the phosphofructokinase and isocitrate dehydrogenase steps. The largest relative reduction was seen in α-ketoglutarate, which was possibly secondary to accumulation of ammonia. There was no change in GABA, but a decrease in glutamate and increases in aspartate and alanine. These, changes are compatible with shifts in the aspartate and alanine aminotransferase reactions, possibly induced by the fall in α-ketoglutarate.     

Measurement of Hippocampal Levels of Cellular Second Messengers Following In Situ Freezing

Abstract: The in situ freezing technique has been widely used to fix labile metabolites and cellular second messengers in cerebral cortex. In this study, we isolated specific brain regions at 0°C from coronal sections of frozen heads following in situ brain freezing and measured regional concentrations of labile metabolites and cellular messengers. These levels in the cortex were compared with those in cortical punches obtained at freezing temperature (less than −40°C) from the same in situ frozen brains and those of cortex dissected from decapitated animals. In both isoflurane- and pentobarbital-anesthetized animals, we observed that the levels of lactate, free fatty acids, inositol 1,4,5-trisphosphate, and diacylglycerol, as well as the proportion of protein kinase C associated with the membrane fraction, were similar in cortical punches taken at freezing temperature and those dissected at 0°C. However, with animals decapitated at room temperature, cortical and hippocampal levels of lactate, free fatty acids, and inositol 1,4,5-trisphosphate and the proportion of membrane protein kinase C were significantly higher than those of corresponding brain regions isolated at 0°C from in situ frozen brains ( p < 0.05). These results indicate that dissection of cortex and hippocampus at 0°C following in situ freezing will eliminate decapitation-induced production of artifacts and changes in the levels of cellular second messengers such as inositol 1,4,5-trisphosphate, diacylglycerol, and protein kinase C. The present technique, used in conjunction with in situ freezing, will fix cellular second messengers and labile metabolites in several regions of brain and may facilitate accurate characterization of molecular and cellular mechanisms underlying CNS function.     

The effect of nembutal on the cellular pulse trains of embryonic nerve tissue transplanted into the brain of the rat

Under nembutal anaesthesia the mean frequency of single unit discharges recorded from allografts of the rat brain was four times lower than the one measured in unanaesthetized animals. A diminished variability was revealed of firing rates and discharge patterns of the cells within grafts in anaesthetized rat brain. In comparison to unanaesthetized animals the number of graft units reacting to a tactile stimulation of contralateral hindlimb of the operated animals was twice lower. Nembutal anaesthesia provoked a full blockade of reactions to the tactile stimuli applied to the ipsilateral side of the animal body.     

31P-NMR studies of cerebral metabolic changes during graded hypoxia in newborn lambs

We measured cerebral phosphocreatine (PCr), inorganic phosphate (Pi), ATP, and intracellular pH (pHi) with in vivo phosphorus nuclear magnetic resonance (NMR) during 10- to 15-min periods of reversible hypoxic hypoxia in 20 newborn lambs (1-11 days). There was a significant correlation between arterial O2 partial pressure (PaO2) and the PCr/Pi ratio or pHi; however, between PaO2 130-33 mmHg, metabolite changes were not significant. PCr/Pi and pHi decreased significantly when PaO2 was lowered below 33 and 28 mmHg, respectively. After recovery, metabolite ratios and pHi returned to base-line values within 5 min. During the early phases of hypoxia and recovery, there were large fluctuations in metabolites and pHi, indicating that mitochondrial reactions were not in a steady state. After several minutes of hypoxia or recovery, PCr/Pi and pHi stabilized, suggesting steady state kinetics for mitochondrial respiration. NMR is extremely sensitive to changes in mitochondrial oxygenation, and stable PCr/Pi and pHi indicate that O2 tension in cerebral mitochondria of the newborn lamb is constant between PaO2 of 30 and 140 mmHg.     

THE EFFECT OF PHENOBARBITONE ANAESTHESIA UPON SOME ORGANIC PHOSPHATES, GLYCOLYTIC METABOLITES AND CITRIC ACID CYCLE-ASSOCIATED INTERMEDIATES OF THE RAT BRAIN

In order to study the effect of phenobarbitone anaesthesia upon the energy metabolism of the brain, organic phosphates, glycolytic metabolites and citric acid cycle intermediates were measured in rats anaesthetized with 175-200 mg/kg of phenobarbitone, and the results were compared to those obtained in rats anaesthetized with halo-thane or with nitrous oxide. An attempt was made to separate the effects of the phenobarbitone anaesthesia from those caused by the accompanying intracellular alkalosis by exposing one group of animals to hypercapnia of such a degree that normalization of the intracellular pH was achieved. Phenobarbitone anaesthesia did not alter the tissue concentrations of ATP, ADP or AMP, but led to a moderate increase in the phosphocreatine concentration. However, since this increase was reversed in the hypercapnic group it is concluded that it may be due partly to a pH-dependent shift in the creatine phosphokinase equilibrium. There was a decrease in the tissue concentrations of all measured substrates from pyruvate and onwards. The results indicate that phenobarbitone leads to a primary inhibition of glycolysis, which cannot be related to detectable changes in ATP, ADP or AMP. The resulting lowering of the tissue concentrations of a number of metabolic acids may be part of the explanation why barbiturate anaesthesia is associated with an intracellular alkalosis.     

Cryogenic 3D printing for tissue engineering

We describe a new cryogenic 3D printing technology for freezing hydrogels, with a potential impact to tissue engineering. We show that complex frozen hydrogel structures can be generated when the 3D object is printed immersed in a liquid coolant (liquid nitrogen), whose upper surface is maintained at the same level as the highest deposited layer of the object. This novel approach ensures that the process of freezing is controlled precisely, and that already printed frozen layers remain at a constant temperature. We describe the device and present results which illustrate the potential of the new technology.     

Changes in cerebral oxygenation and blood flow during LBNP in spinal cord-injured individuals.

Spinal cord-injured (SCI) individuals, having a sympathetic nervous system lesion, experience hypotension during sitting and standing. Surprisingly, they experience few syncopal events. This suggests adaptations in cerebrovascular regulation. Therefore, changes in systemic circulation, cerebral blood flow, and oxygenation in eight SCI individuals were compared with eight able-bodied (AB) individuals. Systemic circulation was manipulated by lower body negative pressure at several levels down to -60 mmHg. At each level, we measured steady-state blood pressure, changes in cerebral blood velocity with transcranial Doppler, and cerebral oxygenation using near-infrared spectroscopy. We found that mean arterial pressure decreased significantly in SCI but not in AB individuals, in accordance with the sympathetic impairment in the SCI group. Cerebral blood flow velocity decreased during orthostatic stress in both groups, but this decrease was significantly greater in SCI individuals. Cerebral oxygenation decreased in both groups, with a tendency to a greater decrease in SCI individuals. Thus present data do not support an advantageous mechanism during orthostatic stress in the cerebrovascular regulation of SCI individuals.     

Effect of Dichloroacetate on Regional Energy Metabolites and Pyruvate Dehydrogenase Activity During Ischemia and Reperfusion in Gerbil Brain

The objective of this study was to determine whether administration of dichloroacetate (DCA), an activator of pyruvate dehydrogenase (PDH), improves recovery of energy metabolites following transient cerebral ischemia. Gerbils were pretreated with DCA, and cerebral ischemia was produced using bilateral carotid artery occlusion for 20 min, followed by reperfusion up to 4 h. DCA had no effect on the accumulation of lactic acid and the decrease in ATP and phosphocreatine (PCr) during the 20-min insult, nor on the recovery of these metabolites measured at 20 and 60 min reperfusion. However, at 4 h reperfusion, levels of ATP and PCr were significantly higher in DCA-treated animals than in controls, as PCr exhibited a secondary decrease in caudate nucleus of control animals. PDH was markedly inhibited at 20 min reperfusion in both groups, but was reactivated to a greater extent in DCA-treated animals at 60 min and 4 h reperfusion. These results demonstrate that DCA had no effect on the initial recovery of metabolites following transient ischemia. However, later in reperfusion, DCA enhanced the postischemic reactivation of PDH and prevented the secondary failure of energy metabolism in caudate nucleus. Thus, inhibition of PDH may limit the recovery of energy metabolism following cerebral ischemia.     

RESTITUTION OF CEREBRAL ENERGY STATE, AS WELL AS OF GLYCOLYTIC METABOLITES, CITRIC ACID CYCLE INTERMEDIATES AND ASSOCIATED AMINO ACIDS AFTER 30 MINUTES OF COMPLETE ISCHEMIA IN RATS ANAESTHETIZED WITH NITROUS OXIDE OR PHENOBARBITAL

Restitution of cerebral cortex concentrations of organic phosphates, glycolytic metabolites, citric acid cycle intermediates, associated amino acids, and ammonia, following a 30 min period of complete ischemia, was studied in rats anaesthetized with either 70% N₂O or 150 mg·kg^-1 of phenobar-bital. Following a 90 min period of recirculation the pattern of restitution was similar in the two groups. Thus, all animals showed recovery of phosphocreatine concentrations, restitution of the adenylate energy charge to about 99% of control, and disappearance of lactate accumulated during the ischemia. Analyses of glycolytic metabolites indicated inhibition of glycolysis at the phosphofructokinase step, possibly caused by accumulation of citrate. Measured citric acid cycle intermediates indicated extensive normalization of mitochondrial metabolism. Changes in amino acid concentrations consisted of a fall in glutamate concentration, a rise in aspartate/glutamate ratio, a fall in GABA concentration, and a rise in alanine concentration. However, ammonia concentration was close to normal, and the size of the amino acid pool did not change. It is concluded that although the results do not exclude damage to a small part of the neuronal population, they demonstrate that, irrespective of the type of anaesthesia used, the majority of brain cells must have survived 30 min of complete ischemia without signs of irreversible metabolic damage.     

Survival of rapidly frozen hatched mouse blastocysts

The objective of the present study was to examine the effect of rapid freezing on the in vitro and in vivo survival of zona-pellucida-free hatched mouse blastocysts. Hatched blastocysts were rapidly frozen in a freezing medium containing either ethylene glycol (EG) or glycerol (G) in 1.5 M or 3 M concentration. Prior to freezing, embryos were equilibrated in the freezing medium for 2 min, 10 min, 20 min or 30 min at room temperature. To freeze them, embryos were held in liquid nitrogen vapour [approximately 1 cm above the surface of the liquid nitrogen (LN2)] for 2 minutes and then immersed into LN2. After thawing, embryos were transferred either to rehydration medium (DPBS + 10% foetal calf serum +0.5 M sucrose) for 10 minutes or rehydrated directly in DPBS supplemented with foetal calf serum. In vitro survival of embryos frozen with EG was higher than those frozen with G. The highest survival was obtained with 3 M EG and 2 min or 10 min equilibration prior to freezing, combined with direct rehydration after thawing. Frozen blastocysts developed into normal foetuses as well as unfrozen control ones did, with averages of 30% (control), 26% (EG) and 15% (G). The results show that hatching and hatched mouse blastocysts can be cryopreserved by a simple rapid freezing protocol in EG without significant loss of viability. Our data indicate that the mechanical protection of the zona pellucida is not needed during freezing in these stages.     

FREE AMINO ACIDS AND RELATED COMPOUNDS IN FIVE REGIONS OF BIOPSIED CAT BRAIN

Abstract— Contents (μmol/g wet wt.) of 34 free amino acids and related compounds were measured in grey matter from three areas of cerebral cortex, from the cerebellum, and from the caudate nucleus in unanaesthetized cats with classical cerveau isolé preparations. Brain specimens were frozen in liquid nitrogen within 10 s of removal; thus, the values found were expected to approximate those which occur in living cat brain. Levels of most of the compounds measured were lower than those previously reported for the cat. In the case of GABA, alanine, and ethanolamine, the lower values found seemed attributable to the rapid freezing of brain tissue, and may more closely approximate levels occurring in living cat brain. On the other hand, the relatively low levels of aspartic and glutamic acids found may have resulted from use of the cerveau isolé preparation. Little difference in levels of amino compounds was found among the three cerebral cortical areas examined. However, there were significant differences in the contents of a number of amino acids between cerebral cortex and the cerebellum or caudate nucleus. These differences resembled those previously observed in autopsied human brain. The content of GABA was two-fold higher in biopsied cat cortex than in biopsied human cortex, whereas the content of cystathionine was only 10 per cent of that in human cortex. Homocarnosine and α-(γ-aminobutyryl)-lysine, two GABA-containing dipeptides found in relatively large amounts in human brain, were not detectable in cat brain. Living cat brain contained two amino acids not previously reported for this species:putreanine and ɛ- N -methyllysine.     

CEREBRAL METABOLIC AND CIRCULATORY CHANGES INDUCED BY HYPOXIA IN STARVED RATS

In order to study cerebral metabolic and circulatory effects of hypoxia under conditions of restricted glucose supply, the arterial P_o2, was reduced to 25–30mm Hg in artificially ventilated and lightly anaesthetized rats that were starved for 24 or 48 h prior to experiments. Arterial glucose concentrations, that were initially around 6μmol g^-1, were significantly reduced after 15min of hypoxia, and decreased to 50^o of control after 30min. In animals studied after 30min of hypoxia (24 h of starvation), cerebral blood flow had increased 4-fold and there was a moderate (25%) rise in cerebral oxygen consumption. During the course of hypoxia, cerebral cortical concentrations of glucose fell to low values. In spite of this, concentrations of pyruvate and lactate rose with time, and the sum of citric acid cycle intermediates (citrate, α-ketoglutarate, fumarate. malate and oxaloacetate) increased. Changes in amino acids were dominated by a fall in aspartate and a rise in alanine concentration. There was a moderate reduction in phosphocreatine and a slight rise in ADP concentration, but concentrations at ATP and AMP were unchanged. The changes observed are similar to those previously obtained in fed animals. It is concluded that even if blood glucose concentrations fall to 3μmol g^-1, and cerebral energy flux is maintained, substrate supply is sufficient to cover the energy requirements of the tissue. Hypoxia was accompanied by increases in the lactate/pyruvate and β-hydroxybutyrate acetoacetate ratios of blood. In the tissue, NADH/NAD⁺ ratios derived from the lactate, malate and β-hydroxybutyrate dehydrogenase systems rose, while that derived from the glutamate dehydrogenase reaction fell. It is concluded that the latter system is not well suited for estimating mitochondrial redox changes in brain tissue.