Library

Notes: —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.

Notes: Abstract— In order to study regulatory steps responsible for the activation of anaerobic glycolysis in the brain during hypoxia, cerebral concentrations of carbohydrate substrates and organic phosphates were measured in rats after reduction of the arterial PO2 to 23-25 mm Hg for 2, 5 and 15 min. The results demonstrated a progressive accumulation of lactate as well as of pyruvate and malate in the absence of changes in ATP, A DP, AMP, citrate and ammonia. The pattern of substrate changes obtained indicate that hypoxia is accompanied by activation of pyruvate kinase and of hexokinase, but not of phosphofructokinase. There was a progressive fall in intracellular pH and a moderate increase in the calculated cytoplasmic NADH/NAD+ ratio. The changes in pyruvate and in the NADH/NAD+ ratio may be responsible for the observed increase in the malate concentration.

Notes: Abstract— In order to evaluate whether porta-caval anastomosis, and the accompanying hyperammonemia, affect the balance between production and utilization of ATP in the brain, organic phosphates and carbohydrate substrates were measured in control and shunted rats exposed to hypoxia (arterial Po2 about 30 mm Hg). In the shunted animals the cortical ammonia content was about 2.5 times that measured in the controls, and there was a marked accumulation of glutamine. The intracellular lactate concentration was identical in the control and the shunted groups, and the pattern of change in carbohydrate substrates was similar. There were no significant differences in ATP, ADP or AMP between the groups but the shunted group showed a significantly lower phosphocreatine content. However, the fall in phosphocreatine in the shunted group could be related to a decrease in the sum of phosphocreatine and creatine. It is concluded that the shunting procedure does not disturb the balance between energy production and energy utilization in the brain.

Notes: Abstract— In order to evaluate the influence of porta-caval anastomosis upon the energy state of the brain, lightly anaesthetized rats were studied either 1 or 5 weeks after the shunting procedure and the brains (frontal lobe, cerebellum and brainstem) were analysed for carbohydrate substrates and organic phosphates. The ammonia contents of arterial blood, cerebrospinal fluid (CSF) and tissue increased progressively in the shunted groups and at 5 weeks the increases were three- to six-fold. In all brain structures studied there were decreases in the glucose and in the aspartate contents but regional differences existed for glucose-6-phosphate, α-ketoglutaratc and glutamate. In the brainstem the tissue contents of glucose-6-phosphate and α-ketoglutarate fell while glutamate was unchanged. Calculation of the cytoplasmatic redox state from the lactate dehydrogenase (LDH) and the malate dehydrogenase (MDH) equilibria indicated that the NADH/NAD+ ratio increased in the shunted groups. However, since there was no significant fall in the calculated adenylate energy charge, it is concluded that porta-caval anastomosis, and the accompanying hyperammonemia, do not disrupt the balance between production and utilization of energy in the brain.

Notes: —The influence of insulin-induced hypoglycemia upon carbohydrate substrates, amino acids and ammonia in the brain was studied in lightly anaesthetized rats, and the changes observed were related to the blood glucose concentration and to the EEG. Calculations from glucose concentrations in tissue, CSF and blood indicated the presence of appreciable amounts of free intracellular glucose at blood glucose concentrations above 3 μmol/g. When the blood glucose concentration fell below 3 μmol/g, there was no calculated intracellular glucose and decreases in the concentrations of glycogen, G-6-P, pyruvate, lactate and of citric acid cycle intermediates were observed. At blood glucose levels of below 1 μmol/g the tissue was virtually depleted of glycogen, G-6-P, pyruvate and lactate.When the blood glucose concentration was reduced below about 2·5 μmol/g there were progressive increases in aspartate and progressive decreases in alanine, GABA, glutamine and glutamate, and at blood glucose concentrations below 2 μmol/g the ammonia concentration increased. It is suggested that most of the changes observed can be explained as a result of a decreased availability of pyruvate and of NADH. The decrease in the concentration of free NADH was reflected in reductions of the lactate/pyruvate and malate/oxaloacetate ratios at an unchanged intracellular pH.Slow wave activity appeared in the EEG when the hypoglycemia gave rise to reduction of the intracellular glucose concentration to zero. Convulsive activity continued until carbohydrate stores in the form of glycogen and G-6-P were depleted. When this occurred the EEG became isoelectric. In all convulsive animals the concentration of the nervous system activity inhibitor, GABA, was decreased and stimulant, aspartate, was increased.

Notes: 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.

Notes: 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.

Notes: Abstract— Optimal freezing conditions for metabolites were evaluated in 250-450 g rats. As a standard procedure, the brains were frozen in such a way that the blood pressure and arterial oxygenation were upheld during the freezing. The progression of the freezing front was determined by means of implanted thermocouples, and the interruption of the circulation by means of injections of carbon particles into the blood stream. The freezing gave rise to a rapid interruption of the circulation in the superficial cortical layer first reached by the freezing front well before the temperature reached 0°C. In deeper regions the progression of the freezing front was slower and interruption of the circulation occurred simultaneously with the freezing of the tissue. Measurements of labile cerebral metabolites, including phosphocreatine, ATP, ADP, AMP and lactate, failed to show signs of autolysis in the part of cortex which became unperfused at temperatures above zero. Since the energy state was identical in superficial cortical areas and in areas that did not freeze until after 40–90 s, it is concluded that the freezing technique gives optimal conditions for metabolites also in deep cerebral structures.Decapitation of unanaesthetized animals gave rise to large autolytic changes in the cerebral cortex. In unanaesthetized animals that were immersed in liquid nitrogen the changes were less marked and mainly affected the concentrations of phosphocreatine, ADP and lactate. When paralysed animals that were anaesthetized with N2O were immersed in liquid nitrogen the only significant change from the control was a decrease in phosphocreatine content. The virtual absence of autolytic changes in this group of animals was not related to the anaesthesia since more pronounced changes were observed in phenobarbitone-anaesthetized rats immersed in the coolant. These differences could be explained by the fact that spontaneously breathing animals immersed in liquid nitrogen developed arterial hypoxia much faster than paralysed animals. It is concluded that an optimal metabolite pattern can only be obtained in anaesthetized animals, frozen with a method that was described by Kerr almost 40 years ago (Kerr, 1935). If unanaesthetized animals must be used, greater attention should be paid to the oxygenation of the blood during the freezing than to such factors as speed of freezing or depth of anaesthesia.

Notes: 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.

Notes: Abstract— In order to evaluate the influence of hypocapnia upon the energy metabolism of the brain, lightly anaesthetized rats were hyperventilated to arterial CO2 tensions of 26, 15 and 10 mm Hg respectively, with subsequent measurements of intracellular pH and of tissue concentrations of carbohydrate substrates, amino acids and organic phosphates. At Pco1= 26 there was a moderate increase in the intracellular pH but when the Pco2 was reduced further to 10 mm Hg the intracellular pH returned to normal, or slightly subnormal, values. The reduction in PCo2 was accompanied by increased cerebral cortical concentrations of lactate, pyruvate, citrate, α-ketoglutarate, malate and glutamate and by decreased aspartate concentrations. It is concluded that the accumulation of metabolic acids explains the normal value for intracellular pH at very low CO2 tensions. Previous results obtained in man indicate that there is an increased anaerobic production of lactic acid in the brain in extreme hypocapnia. At comparable CO2 tensions the present results showed a small fall in phosphocreatine and a small rise in ADP. However, since the ammonia concentrations were normal or decreased and since there was an increase in citrate, the results give no direct support to the hypothesis of an activation of phosphofructokinase. Since the cerebral venous Po2 was reduced to 20 mm Hg at an arterial CO2 tension of 10 mm Hg the accumulation of acids was probably secondary to tissue hypoxia. However, since there was no, or only a very small, increase in the calculated cytoplasmic NADH/NAD+ ratio, it appears less likely that acids accumulated due to lack of NAD+.