Effect of global vs regional ischaemia upon myocardial contractility and oxygen balance.

The possible role of catecholamines upon the nature of myocardial response to regional and global left ventricular ischaemia was investigated. Global ischaemia was accomplished by temporary occlusion of the left main coronary artery and regional ischaemia by temporary ligation of the left anterior descending coronary artery. Isometric force of contraction was measured with strain gauge arches, regional blood flow by a thermistor technique and intracellular NADH redox level by a fluorometric technique. These measurements were made simultaneously in two areas on the left ventricular myocardium: one immediately below the bifurcation of the LAD and the other immediately below the circumflex coronary artery. Following LAD occlusion a variable inotropic response was observed in the ischaemic area. A decrease in contractile tension was found in 19 of the dogs (44%), no change in 23% and an increase was found in 33%. Global ischaemia invariably resulted in a decrease in contractile tension. NADH redox state was markedly higher during global than during regional ischaemia. Propranolol administration blocked the positive inotropic response to regional ischaemia. It is concluded that endogenous catecholamine release may be responsible for increased contractile force during regional ischaemia. This response is apparently limited by the oxygen availability to the tissue.

Relationship between local oxygen consumption and local and external cardiac work: effect of tachycardia.

This study was designed to determine the extent of coupling between regional myocardial segment work and corresponding regional oxygen consumption, and to examine whether tachycardia induced changes in regional work are translated into corresponding changes in external cardiac work. In the open chest anaesthetised dog, the heart was paced at frequencies of 120-270 beats.min-1. Global and regional myocardial O2 supply, consumption, and balance were evaluated at each heart rate, and correlated with corresponding functional changes. Global cardiac function was evaluated from aortic flow, blood pressure, and left ventricular pressure. Coronary sinus flow and O2 saturation were used to calculate O2 consumption. The integrated multiple of myocardial shortening (ultrasonic dimension crystals) by corresponding force (strain gauge arch) during an averaged beat was used to express regional segment work. Regional coronary blood flow was measured with radioactive microspheres, and microspectrophotometry was used to evaluate O2 saturation in small arteries and veins. These indices were used to calculate regional myocardial oxygen consumption. NADH redox levels were recorded by surface fluorometry, and were found to increase with heart rate by up to 67%. Increasing heart rate from 120 to 180 beats.min-1 increased regional work from 3040(SEM 220) to a peak of 4290(280) mm.g-1.min-1, whereas external cardiac work did not increase [67.0(2.6) to 65.3(4.4) mm Hg.litre-1.min-1] and fell further at the highest rates. Regional oxygen consumption increased from 6.16(0.47) to 8.29(0.53) ml O2.min-1.100 g-1 and was linearly related to regional work at all heart rates (r = 0.971, p less than 0.05). External cardiac work fell by about 26% whereas global myocardial oxygen consumption increased by 49% during tachycardia. It is concluded that myocardial oxygen consumption is more closely related to regional segment work than to external work, and that tachycardia significantly raises the oxygen cost of external work of the heart.

Relationship between intracranial pressure and cortical spreading depression following fluid percussion brain injury in rats.

Traumatic brain injury (TBI) is known to be accompanied by an increase in intracranial pressure (ICP) and in some cases, by spontaneous generation of cortical spreading depression (CSD) cycles. However, the role of CSD in the pathophysiology of cerebral contusion is still unknown. A multiparametric monitoring assembly was placed on the right hemisphere of the rat brain to evaluate ICP, DC potential, extracellular K(+), cerebral blood flow (CBF), and electrocorticogram in 27 rats during 5 h. Fluid percussion brain injury (FPBI) with the magnitude of the impact 2.9, 3.3, 4.1, and 5.0 atmospheres was induced to the left parietal cortex in animal groups A, B, C, and D, respectively. A slow increase in ICP was evident, and was pronounced in group C and especially in group D, where four of nine animals died during the monitoring. At the end of the 5 h experiment, the mean ICP levels were 6.75 +/- 2.87, 8.40 +/- 2.70, 12.75 +/- 4.03, 29.56 +/- 9.25, and the mean total number of CSD cycles was 2.00 +/- 1.41, 4.29 +/- 4.23, 11.71 +/- 13.29, and 20.11 +/- 19.26 in groups A, B, C, and D, respectively. The maximal level of intensity of CSD cycle generation after FPBI was obtained in group D, where almost constant activity was maintained until the end of the experiment. A significant coefficient of correlation between ICP level and total number of CSD cycles was found for all ICP measurements (r = 0.47-0.63, p < 0.05, n = 27), however more significant (p < 0.001) was the coefficient during the period of monitoring between 2 and 4 h after FPBI. Our results suggest that numerous repeating CSD cycles are typical phenomena in moderate and especially severe forms of FPBI. The rising number of CSD cycles under condition of an ICP level >/=20 mm Hg may demonstrate, with high probability, the unfavorable development of TBI, caused by growing secondary hypoxic insult.

Regulation of chondrogenic differentiation of mesenchymes by protein kinase C alpha.

Chondrogenesis of chick limb bud mesenchymes requires the expression and activation of protein kinase C (PKC). This study was performed to identify PKC isoform(s) involved in the regulation chondrogenic differentiation of mesenchymes. Multiple PKC isoforms including alpha, epsilon, zeta and lambda/iota were expressed in mesenchymes derived from chick limb buds. Among the expressed PKC isoforms, the levels of PKC alpha and epsilon were increased during chondrogenic differentiation of mesenchymes. The increase in the expression of these isoforms is more evident in the particulate membrane fraction compared with the cytosolic fraction. Chondrogenesis was blocked by either selective inhibition or down-regulation of PKC alpha. In addition, the degree of chondrogenesis was closely correlated with the expression levels of PKC alpha but not other PKC isoforms expressed in mesenchymes. Thus, the results indicate that only PKC alpha is required for the induction of chondrogenic differentiation

The effect of ethanol on metabolic, hemodynamic and electrical responses to cortical spreading depression.

Alcohol induces a decrease in cerebral blood flow (CBF) and metabolic rate, mitochondrial damage and other impairments in brain function and structure. Cortical spreading depression (CSD) is a phenomenon causing changes in ion homeostasis and raises energy demand, mitochondrial activity and CBF. It is of great interest to study the effect of ethanol on brain response under a challenge of increasing oxygen demand by inducing CSD. A special multisite assembly (MSA) was constructed to evaluate metabolic (mitochondrial NADH), hemodynamic (reflectance) and electrical (DC potential) activities from four parasagittally adjacently arranged areas of the cerebral cortex, continuously and simultaneously in vivo. Three CSD cycles were initiated every 30 min before and after ethanol or saline infusion over 4.5 h. During CSD amplitude changes of reflectance, NADH and DC potential as well as propagation rates and wave frequency were calculated. After ethanol infusion CSD showed a decrease in the negative shift of the DC potential, and alterations in the biphasic responses in reflectance, which may indicate alteration in blood volume: unclear responses in the initial vasoconstriction phase and a significant increase in the subsequent vasodilatation phase. The reduction in the amplitude of the NADH oxidation cycle may depict a decrease in energy production, which could also be indicated by a decline in wave frequency (prolonging the recovery phase of the CSD). The decrease in propagation rate indicates a decline in tissue excitability and in the CSD initiation mechanism induced by ethanol treatment.

Effects of brain oxygenation on metabolic, hemodynamic, ionic and electrical responses to spreading depression in the rat.

The effect of cortical spreading depression (CSD) on oxygen demand (extracellular K(+)), oxygen supply (cerebral blood flow - CBF) and oxygen balance (mitochondrial NADH) was studied by a special multiprobe assembly (MPA), during hypoxia and partial ischemia. The MPA was constructed and applied to monitor the CSD wave from its front line until complete recovery, continuously and simultaneously. CSD under hypoxia or partial ischemia led to an initial increase in NADH levels and a further decrease in CBF during the first phase of the CSD wave, indicating a decrease of tissue capability to compensate for an increase in oxygen demand. Furthermore, the special design of the MPA enabled identifying the close interrelation between oxygen demand, supply and balance during CSD propagation. In conclusion, brain oxygenation was shown to have a clear effect on tissue responses to CSD.

Responses of rat brain to induced spreading depression following exposure to carbon monoxide.

Until recently carbon monoxide (CO) was known only for its noxious effects. Exposure to CO results in an autoregulatory increase in cerebral blood flow (CBF). Little information is available on brain energy metabolism under low CO concentrations and on the effect of CO on the stimulated brain. In this study cortical spreading depression (SD) was induced in order to cause transient brain depolarization and increased energy demand. The multisite assembly (MSA), which contains four bundles of optical fibers for monitoring the intramitochondrial NADH redox state and tissue reflectance as well as four DC electrodes enabling measurement from four consecutive points on the cerebral cortex, was used to measure energy metabolism and the propagation of SD waves during exposure to CO. CBF in the contralateral hemisphere was measured using the laser Doppler technique. Three experimental groups of animals were examined: SD was induced during exposure to 1000 ppm CO, immediately after exposure to CO and 90 min after cessation of exposure to CO. Three control groups were also examined, in which the animals underwent the same procedures but were not exposed to CO. In all animals exposure to CO was followed by a significant increase in CBF. The greatest effect was found when SD was induced immediately after cessation of exposure to CO. SD wave frequency decreased when induced immediately after exposure to CO, whereas it increased when SD was induced 90 min after exposure. The amplitude of the NADH oxidation waves and their integral were smaller during SD induced immediately after exposure to CO. The DC potential did not change, suggesting that CO did not affect the SD initiation mechanism but rather resulted in energy depletion during recovery from SD. This study demonstrates that even at a concentration of 1000 ppm CO interferes with the metabolic activity of the brain during repolarization of the SD-induced negativity.

Multiparametric monitoring of brain oxygen balance under experimental and clinical conditions.

In order to evaluate the relationship between brain oxygen supply and demand (O2 balance) in real time, it is necessary to use a multiparametric monitoring approach. Cerebral blood flow (CBF) is a representative parameter of O2 supply. The extracellular level of K+ is a reliable indicator of O2 demand since more than 60% of the energy consumed by the brain is utilized by active transport processes. Mitochondrial NADH redox state can represent the balance between O2 supply and demand. In order to monitor the brain of experimental animals or patients, we constructed the multiparametric assembly (MPA) and the following parameters were monitored simultaneously and in real time: CBF, CBV, NADH redox state, extracellular K+, DC potential, EEG, tissue temperature and ICP. Animals were exposed to hypoxia, ischemia, hypercapnia, hyperoxia and spreading depression (SD) and the relative changes in CBF and NADH were calculated and found to be significant indicators of brain energy state. Monitoring these two parameters increases the possibility of differentiating between various pathophysiological states. Each added parameter increases the power of diagnosis and determination of the functional state of the brain. Preliminary results obtained in patients monitored in the ICU or in the OR show that the responses to hypercapnia, spreading depression or ischemia are similar to those measured in experimental animals.

Fiber optic surface fluorometry-reflectometry technique in the renal physiology of rats.

Most current knowledge on events in the mitochondria leading to acute renal failure originates from studies in which indirect methods were used. The disadvantage of these methods is that they cannot measure the turnover rate of various metabolites, and only one result per animal can be obtained. Chance et al. /9/ developed a method using optical techniques for continuously monitoring the fluorescence of intramitochondrial NADH, which has been applied mainly to the brain. This optical method has not yet been examined quantitatively in the kidney and no attempt has been made to adapt this method for routine measurement in kidney tissue. The purpose of this study was to adapt the surface fluorometry method for monitoring renal NADH redox state in situ, and to determine whether the hemodynamic artifacts involved in fluorometric studies of the renal surface in situ could be eliminated by using a correction factor. Another purpose was to understand the relationship between the changes in reflectance and blood volume in the rat kidney. This was achieved by measuring the reflectance after: a) blood exchange by FC-43 emulsion; b) intrarenal saline flush; c) occlusion of the renal vein, renal artery and reopening of the renal vein; d) calculation of the correlation between changes in kidney weight after renal artery occlusion, and the reflectance. Our results suggest that in the rat kidney, as opposed to the brain, a correction factor of 1:1 is not always applicable. This factor may vary between animals, and it is therefore necessary to adjust it electronically for each rat kidney. This observation contradicts the view suggesting a constant correction factor of 1:1 in the kidney. The results reported herein indicate that changes in the reflectance in the ischemic rat kidney are due to changes in blood volume. In conclusion, it seems that optical techniques for monitoring fluorescence are suitable for localized, continuous and non-invasive recording of tissue mitochondrial NADH redox states under various conditions in the rat kidney.

Responses to Cortical Spreading Depression under Oxygen Deficiency.

OBJECTIVES: The effect of cortical spreading depression (CSD) on extracellular K(+) concentrations ([K(+)](e)), cerebral blood flow (CBF), mitochondrial NADH redox state and direct current (DC) potential was studied during normoxia and three pathological conditions: hypoxia, after NOS inhibition by L-NAME and partial ischemia. METHODS: A SPECIAL DEVICE (MPA) WAS USED FOR MONITORING CSD WAVE PROPAGATION, CONTAINING: mitochondrial NADH redox state and reflected light, by a fluorometry technique; DC potential by Ag/AgCl electrodes; CBF by laser Doppler flowmetry; and [K(+)](e) by a mini-electrode. RESULTS AND DISCUSSION: CSD under the 3 pathological conditions caused an initial increase in NADH and a further decrease in CBF during the first phase of CSD, indicating an imbalance between oxygen supply and demand as a result of the increase in oxygen requirements. The hyperperfusion phase in CBF was significantly reduced during hypoxia and ischemia showing a further decline in oxygen supply during CSD. CSD wave duration increased during the pathological conditions, showing a disturbance in energy production.Extracellular K(+) levels during CSD, increased to identical levels during normoxia and during the three pathological groups, indicating correspondingly increase in oxygen demand. 5. The special design of the MPA enabled identifying differences in the simultaneous responses of the measured parameters, which may indicate changes in the interrelation between oxygen demand, oxygen supply and oxygen balance during CSD propagation, under the conditions tested. 6. In conclusion, brain oxygenation was found to be a critical factor in the responses of the brain to CSD.

Activity of protein kinase C during the differentiation of chick limb bud mesenchymal cells.

To investigate the relationship between protein kinase C (PKC) and chondrogenesis, PKC activity was assayed in cultures of stage 23/24 chick limb bud mesenchymal cells under various conditions. PKC activities of cytosolic and particulate fractions were low in 1 day cultured cells. As chondrogenesis proceeds, cytosolic PKC activity increased more than twofold, while that of the particulate fraction increased only slightly. Three days' treatment of cultures with phorbol-12-myristate-13-acetate (PMA, 5 x 10(-8) M) inhibited chondrogenesis judged by the accumulation of Alcian blue bound to the extracellular matrix and depressed PKC activity in cytosolic fraction. When cells were grown for 3 days in control medium after 3 days' treatment with PMA, chondrogenesis resumed and PKC activity recovered to normal values. PKC activity in cultures plated at low density (5 x 10(6) cells/ml) where chondrogenesis is reduced was as low as that in 1 day cultured cells plated at high density (2 x 10(7) cells/ml) or that in PMA treated cells. On the other hand, staurosporine promoted chondrogenesis without affecting PKC activity. Furthermore, reversal of PMA's inhibitory effect on chondrogenesis by staurosporine was not accompanied by recovery of PKC activity. These data indicate that increases in PKC activity is closely related to chondrogenesis and that PMA inhibits chondrogenesis by depressing PKC. However, staurosporine's enhancing effect on chondrogenesis is not related to PKC activity.

Na+-Ca2+ exchange in regulation of contractility in canine cardiac Purkinje fibers.

To study Na+-Ca2+ exchange, intracellular Na+ activity (aiNa), twitch tension, and transmembrane potential were simultaneously measured in canine cardiac Purkinje fibers driven at a constant rate (1 Hz) in the absence and presence of strophanthidin (5 X 10(-7) M) at normal, low, and high extracellular [Na+] ([Na+]o) or [Ca2+] ([Ca2+]o). Intracellular Ca2+ activity (aiCa) of the fibers was also measured in a normal Tyrode solution. Reductions of [Na+]o by 20, 40, and 60% decreased the ratio of extracellular Na+ activity (aoNa) and aiNa in the steady state but steeply increased twitch tension. This finding is consistent with the view that a decrease in aoNa/aiNa increases intracellular Ca2+ through Na+-Ca2+ exchange. In further agreement with this view, a Na+-free solution virtually depleted intracellular Na+ and increased the resting tension of the fibers. The slope of the relation of the logs of twitch tension and aiNa that was determined at normal [Na+]o and [Ca2+]o may reflect the properties of the Na+-Ca2+ exchange. Slope of log tension-aiNa relationship decreased when reducing [Na+]o or increasing [Ca2+]o had decreased the level of aiNa. On the other hand, the slope increased when a rise in [Na+]o or a reduction in [Ca2+]o had increased the level of aiNa. These results indicate that as the aiNa level increased, slope of tension-aiNa relation increased, which suggests that Na+-Ca2+ exchange may depend on level of aiNa.(ABSTRACT TRUNCATED AT 250 WORDS)

An experimental approach for evaluation of the O2 balance in local myocardial regions in vivo.

Quantitative evaluation of myocardial oxygen balance can be accomplished by measurement of oxygen supply, demand, and intracellular oxygen concentration. Experimentally, these parameters are often related to coronary blood flow, cardiac contractility, and mitochondrial NADH redox level, respectively. Methods were developed to measure these three parameters in a local region on the myocardial surface in open-chest dogs. Local coronary blood supply was measured with the aid of a small surface thermistor, and cardiac work with a miniature strain gauge arch. NADH oxidation--reduction state was recorded using surface fluorometry through a fibre optic light guide. Transient anoxia produced by nitrogen breathing caused a rapid but reversible elevation in NADH levels, which was not always accompanied by a concomitant change in contractile force. Elevation of heart rate resulted in a rise in intramitochondrial NADH followed by an increase in coronary flow. In spite of the increased flow, NADH levels remained elevated, indicating a change in the O2 balance; this may indicate that autoregulation does not necessarily result in total compensation.

Dynamic redistribution of coronary blood flow in the dog as measured by a thermistor technique.

The effect of heart rate upon the coronary blood supply to the right and left ventricles was studied in anaesthetized, open-chest dogs. Indirect recording of coronary supply was obtained using thermistors placed locally on the surface or in the wall of the ventricles. The validity of the thermistor technique was examined in vivo as well as by using an in vitro laboratory model. Myocardial perfusion was also measured using electromagnetic flowmeters placed around the anterior descending and circumflex arteries, as well as the coronary sinus and Thebesian drainage. It was found that at heart rates lower than about 150/min, coronary perfusion to all areas increased equally as rate was elevated. At higher heart rates, coronary flow to the right ventricle continued to increase linearly with rate, whereas perfusion to the left ventricle either increased much less or declined. It is concluded that tachycardia causes coronary flow to be dynamically redistributed in favour of the right ventricle.

Effect of coronary vasodilation produced by hypopnea upon regional myocardial oxygen balance.

An attempt was made to differentiate between autoregulatory coronary vasodilation and changes in vasomotor tone produced by factors extrinsic to the heart. this was done by investigating the relation between local cardiac force and local coronary blood supply. Intracellular NADH redox levels were also measured in order to further elucidate the oxygen balance under various experimental conditions. In anaesthetized open-chest dogs, local blood supply was estimated with the aid of a thermistor probe, and the oxidation-reduction state of mitochondrial pyridine nucleotide was measured by a surface fluorometric technique. Local myocardial contractile force, as well as blood pressure and ECG were recorded simultaneously with the above parameters. The heart was paced at frequencies from 60/min to 300/min with an electronic stimulator, under both normoxic and hypopneic conditions. It was found that elevation of heart rate caused a progressive increase in local blood flow during both normal and hypopneic ventilation. The absolute flow values during hypopnea were approximately double those during normoxia. Heart rates above 120/min or 150/min resulted in a progressive increase in NADH fluorescence. This response to elevated heart rate was less prominent or absent during hypopnea. Contractile force during hypopnea was greater at elevated heart rates than during normal breathing. Data are brought which suggest that whereas vasodilation following increased heart rate is probably due to an autoregulatory mechanism, the marked vasodilatatory effect of hypopnea is related to elevated arterial CO2 levels. It is suggested that hypercapnia markedly stimulates extrinsic coronary vasodilation thereby supplying enough oxygen to maintain contractility even at very high heart rates. Moreover, intracellular O2 concentration (mitochondrial NADH level) is maintained at a normal level despite the greatly increased demand.

Coronary vasodilation produced by tachycardia under various basal flow conditions.

Some properties of coronary vasodilation produced by heart rate elevation under various basal coronary flow levels was studied. Coronary sinus blood flow, myocardial oxygen consumption and left ventricular contractile force were measured in anaesthetized, open-chest dogs. Heart rate was progressively increased by electrical stimulation at rates ranging from 60/min to 210/min. This was repeated during control, noradrenaline infusion (0.2 microgram kg-1 min-1), in the presence of propranolol (0.25 mg/kg), and during hypopneic positive pressure respiration. It was found that under all experimental conditions, coronary perfusion increased linearly with heart rate. At each rate, coronary flow was greater during noradrenaline infusion and hypopneic respiration than that observed during control or following beta-blockade. Myocardial oxygen consumption behaved similarly to flow, and MVO2 was lowest in the presence of propranolol, and highest during hypopneic ventilation and catecholamine infusion. Contractile force per min (heart rate x tension) also increased with increasing heart rate, but was greatest during noradrenaline infusion, lowest during beta-blockade, and similar during both control and hypopneic respiration. These results indicate that the oxygen cost of contraction was different under the various conditions, and was particularly wasteful during hypopneic respiration. It is concluded that autoregulation caused by heart rate elevation is not dependent on the initial state of coronary blood flow, and that endogenous catecholamine release cannot account for this phenomenon.