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.     

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.     

Inflation reflex in the rat.

The Breuer-Hering inflation reflex [BHIR] was elicited in conscious and anaesthetized rats by inflating the lungs with constant pressures of 5--20 cm H2O. The reflex was elicited well in conscious animals, but even with the maximum stimulus [inflation of 20 cm H2O, corresponding to about 4.5-fold the tidal volume] the duration of apnoea did not exceed 4 control respiration cycles. In anaesthetized animals, the same stimulus let to apnoea lasting 180--400 control respiration cycles on the average, according to the type and depth of general anaesthesia. The duration of apnoea in occlusion of the air passages in the expiratory position increased with the depth of anaesthesia, while in occlusion of the air passages at the peak of inspiration it was shortened. Stimulation of chemoreceptors [inhalation of a mixture 4% CO2 in O2 or of 8% O2 in N2] did not influence the elicitability or duration of the BHIR, nor did cooling the rats to 28 degrees C or heating them to 38 degrees C. The mean respiration frequency was 98 c/min in unanaesthetized rats, 96 c/min in urethane anaesthesis and 79--48 c/min in halothane anaesthesia, according to the depth of anaesthesia. Bilateral cervical vagotomy reduced mean respiration frequency to 35.6 c/min in conscious rats and to 31 c/min in urethane-anaesthetized animals. The results indicate the existence of species-related differences between basic regulatory mechanisms in the rat and certain other mammals.     

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.     

OPTIMAL FREEZING CONDITIONS FOR CEREBRAL METABOLITES IN RATS

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.     

The effects of altered endocrine states and of ether anaesthesia on mouse brain

The effects of ether anaesthesia on metabolites of mouse brain in altered endocrine states has been examined. Alloxan diabetic mice, with elevated levels of blood and brain glucose, exhibited changes in brain metabolites after ether anaesthesia that were comparable to those seen in normal animals. Sympathectomized and/or adrenalectomized mice had decreased levels of brain glucose. The percentage elevation of glucose in the brains of these animals under ether anaesthesia approximated to normal values, although the absolute cerebral levels were lower. Increases in glycogen in the brains of these animals were somewhat diminished. In none of the altered endocrine states were the changes in brain metabolites following ether anaesthesia eliminated. The activity of UDPglucose-glycogen glucosyltransferase (UDPglucose: glycogen α-4-glucosyltransferasee, EC 2.4.1.11) in the mouse brain was measured in the absence and in the presence of glucose-6-P. Neither the total activity nor the percentage of the I form (measured in the absence of glucose-6-P) was altered by anaesthesia or by the endocrine state of the animal. The Michaelis constants with UDPglucose as substrate for the total and I forms were 0·36 mM and 1·0 mM, respectively. Considerable UDPglucose-glycogen glucosyltransferase activity was observed in the absence of added glycogen primer. The observed increase in activity in the presence of added glucose-6-P was greater than would have been anticipated if the hexose phosphate were acting at only one site.     

Influence of cold and warm acclimation on neuronal responses in the lower brain stem

Neurons in two lower brain stem areas, the nucleus raphe magnus and the subcoeruleus region, have been shown to be part of the thermoafferent system. It is concluded from microcut experiments in unanaesthetized guinea pigs that inhibition of shivering caused by nucleus raphe magnus stimulation is mediated partly by ascending and partly by descending efferents of the nucleus raphe magnus. Electrical stimulation of the subcoeruleus area caused excitatory metabolic responses. Interruption of the ascending efferents of the subcoeruleus area did not prevent the metabolic activation. It is concluded that the excitatory responses are partly mediated by descending efferents of the subcoeruleus area. The descending pathways project mainly to motoneurone pools and to dorsal horn cells. In cold-acclimated guinea pigs, the average maximum activity of bell-shaped subcoeruleus cold-responsive units was reduced significantly in comparison with cold-responsive neurons in animals acclimated to normal room temperature. Furthermore, peak activity of warm-responsive units in the nucleus raphe magnus was larger in cold-acclimated animals than in animals acclimated to normal room temperature. These neuronal changes may contribute via descending lower loops and via ascending upper loops to long-term slope reduction of metabolic cold defence and shivering threshold displacements.     

Spontaneous activity of olfactory bulb neurons in awake rabbits, with some observations on the effects of pentobarbital anaesthesia

A specially adapted microelectrode driver device has been used to record the spontaneous activity of neurons in the olfactory bulb of awake rabbits. Several parameters of this activity were studied in 78 neurons of conscious animals. A second experiment was performed to investigate anaesthetic-induced modifications of the spike discharge initially recorded in awake animals. 1. In unanaesthetized animals, the interspike interval distribution of all cells was found to be stable over short as well as long periods of time. 2. A periodical change in firing probability, correlated with respiratory activity, was observed in a high percentage of neurons. During inspiration, the discharge was markedly increased ("well synchronized" neurons, n = 2), slightly increased ("poorly synchronized" neurons, n = 15); or unchanged ("not synchronized" neurons, n = 8). 3. The passage from the awake to the anaesthetized state resulted in more regular cell activity with sudden changes from one steady firing level to another, without affecting the cell classification. As anaesthesia wore off, the cell units recovered the characteristic discharge pattern initially observed.     

PGF2 alpha and breathing pattern in newborn pigs

The effect of PGF2 alpha has been evaluated in 11 unanaesthetized unrestrained piglets and in 3 anaesthetized piglets (2-3 days old) using a barometric-plethysmographic technique. PGF2 alpha (mg 0.25/pig) was administered as aerosol for 5 min. In 3 of the unanaesthetized newborn pigs the effect of PGF2 alpha aerosol has been evaluated after indomethacin (mg 1/Kg i.v.). The vagal dependent activity of the prostaglandin was also evaluated after atropine (mg 0.08/Kg i.m.). Our results show that PGF2 alpha in newborn pigs causes hypoventilation due to a decrease in respiratory rate and to a lengthening in TE. The changes in TE are due to an increase in the incidence and duration of apneic events characterizing the respiratory activity at birth. After indomethacin PGF2 alpha does not change the breathing pattern. Atropine only partially reduces the effects of PGF2 alpha while, after anaesthesia, prostaglandin does not change the breathing pattern. Consequently our results show that PGF2 alpha in newborn animals similar to other prostaglandins acts as a depressant of respiratory activity.     

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.     

The absorptive viability of isolated intestine prepared from dead animals.

Glucose and water absorption rates are much lower (50%) in isolated perfused rat small intestine when the animals are killed by stunning or ether before removal of the intestine than when the animal is maintained alive under light ether anaesthesia throughout the setting-up procedure.     

Effect of thyrotropin releasing factor on body weight of the pond snail Lymnaea stagnalis.

Earlier studies had demonstrated that in Lymnaea stagnalis thyrotropin releasing factor (TRF) may be the secretory product of the so-called dark green neurosecretory cells. The dark green cells are believed to serve an osmoregulatory function. If TRF is the secretory product of the dark green cells, it should be capable of controlling the salt and water balance in L. stagnalis. In this study, the effect and fate of synthetic TRF injected in vivo into L. stagnalis was assessed. It was found that TRF caused an increase in the rate of loss of body water which normally occurs after anaesthesia. TRF also increased the loss of body water when it was administered to unanaesthetized animals. The peptide was accumulated and degraded by the tissues of the foot, mantle, and head regions, tissues which are believed to be the targets of the hormone of the dark green cells. Our results support the hypothesis that TRF may be the secretory product of the dark green cells and may be involved in osmoregulation in L. stagnalis.     

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.     

Biochemical studies on histones of the central nervous system. III. Incorporation of [14C]-acetate into the histones of different rat brain regions during a learning experiment

Long term memory formation obviously is accompanied by changes in macromolecule synthesis within the brain structures involved. The regulation of this process was investigated in various rat brain regions using the model of histone acetylation with [14C]-acetate during the training session, as well as 5 min and 120 min upon completion of training. Almost at all three times and in most brain structures the acetylation rate was diminished in trained animals as compared to pseudo-conditioned rats. With the exception of hippocampus labeling in trained animals was even lower in comparison to passive controls.     

A mechanical device for the rapid removal and freezing of liver or brain tissue from unanaesthetized and nonparalyzed rats.

A mechanical, pneumatically operated device has been developed that permits withdrawal and freezeclamping of either liver or brain tissue from unanaesthetized nonimmobilized rats. Furthermore, regional sampling from brain is possible. The apparatus works in the following way: By means of rotating knives, two cross-sections are made through a rat in such a way that the slice cut out of the animal between the two knives will contain the desired tissue. This slice is then freezeclamped between precooled aluminium blocks. The sampling procedure works automatically: i.e., when the rat is in position in the apparatus and the precooled aluminium blocks have been mounted on the arms of a pair of cooling tongs, the biopsy is made simply by pressing a button. The time for making a biopsy amounts to 0.10 sec. The brain or liver tissue can be isolated from the freezeclamped slices at the temperature of liquid N₂, using a small buzz saw developed for this purpose.     

Estrone sulfate sulfohydrolase in the developing brain and pituitary of rat, mouse and guinea pig

The pattern of estrone sulfate sulfohydrolase (estrogen sulfatase) development in the brain of rat, mouse and guinea pig has been established by assaying whole homogenates. Activity was measurable in each species from the fetal state to adulthood. Maximum brain content was reached at about 20 days of age in rat, 14 days in mouse and 15 days in guinea pig. A considerable decrease occurred between 14 days and adulthood in mouse and lesser decreases were seen in rat and guinea pig. The subcellular distribution of enzyme in rat and mouse brain appeared to change from the immature to the adult state. No major differences in enzyme activity occurred between the sexes at any age. Tissue concentration of enzyme in the hypothalamic-preoptic area of rat and mouse was similar to that in the remainder of the brain. In guinea pig the brain concentration was slightly lower than that of the hypothalamic-preoptic region. Sulfatase content of the pituitary was low in all 3 species but the tissue concentration was considerably higher than that of brain, particularly in rat and mouse. Apparent Km values for brain sulfatase were in the range 6-17 microM, with no striking sex difference. Apparent Km's for pituitary sulfatase of immature rat and guinea pig were similar to those for brain in the same animals but that for mouse pituitary (0.9 microM) was much lower. It is unlikely that brain or pituitary sulfatase is by itself, a major factor in making available potentially active estrogen for use during differential sex development in these species.     

Effect of antenatal ethanol exposures on the function of the hemato-encephalic barrier in animals

Blood-brain barrier permeability for P32, H3-valine, H3-tyrosine, S35-methionine was investigated in rat offsprings, 20 and 60 days old, that had been antenatally exposed to ethanol. In experimental 20-day-old rat offsprings P32 incorporation in brain structures was lower than in the controls. In mature rats that had been exposed to antenatal ethanol, a different degree decrease in labelled amino acid penetration and alterations in P32 penetration into different brain structures were found. Significant differences in stress-induced changes of blood-brain barrier permeability were observed in experimental and control animals.     

Measurement of in vivo incorporation of L-methionine-35S into cerebral proteins by quantitative autoradiography in freely-moving or anesthetized rats

Incorporation of L-35S-methionine in brain proteins was measured by quantitative autoradiography in the rat. This model was checked by biochemical procedures. In awake freely moving rat, methionine incorporation rates vary from 36 to 258 nmol/100g/min. During anaesthesia, protein synthesis in brain decreases (30 to 50%).     

Neuronal responses of the rate somatosensory cortex transplanted into the vibrissae representation field of the neocortex to the electrical stimulation of the recipient brain

Neuronal responses of the rat somatosensory cortex grafted into damaged host barrel field to electrical stimulation of the host brain were investigated extracellularly in rats under light pentobarbital anaesthesia. The following structures of the host brain were stimulated: ventrobasal complex and posterior thalamic nuclei, ipsilateral area of vibrissae representation in the sensorimotor cortex and contralateral barrel field. Reactivity of the grafted neurones was lower, than in the intact barrel field, but the mean latencies of responses were not significantly different. Stimulation of the thalamic nuclei was more effective than that of the cortical areas both in grafted and intact barrel fields. Posttetanic depression after repetitive stimulation was often observed in the grafts, while posttetanic potentiation was more usual for the intact barrel field. The data show the sources of some functional afferent inputs to the grafts which may be responsible for neuronal reactions to somatosensory stimulation of the host animal.     

Stabilization of the colchicine-binding activity of tubulin by glutathione and by dietary selenium-deficiency

The lability of tubulin in supernatant fluids from rat brain was studied by measuring the loss of colchicine-binding activity. During incubation at 37 degrees C for 1 hr approximately 50% of the binding activity of rat brain supernatant fluids was lost. This loss was reduced by mercaptoethanol or reduced glutathione. Anaerobic conditions did not affect loss of binding, while oxygen markedly reduced it, especially in the presence of reduced glutathione. Supernatant fluids prepared from brains of animals deficient in selenium showed a lower loss of binding than those from selenium-supplemented controls.