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.

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.

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.

Disruption of actin cytoskeleton induces chondrogenesis of mesenchymal cells by activating protein kinase C-alpha signaling.

Disruption of actin cytoskeleton with cytochalasin D has been known to induce chondrogenic differentiation of chick embryo limb bud mesenchymal cells. However, the mechanism(s) for the induction of chondrogenesis by cytochalasin D is not yet clearly known. In the present study, we examined possible involvement of protein kinase C (PKC) and extracellular signal-regulated protein kinase (Erk-1) in chondrogenesis of mesenchymal cells induced by disruption of actin cytoskeleton. Disruption of actin cytoskeleton with cytochalasin D or latrunculin B induced chondrogenesis of mesenchymal cells cultured at subconfluent cell density, as determined by type II collagen expression. Among the expressed PKC isoforms, cytochalasin D dramatically increased expression and activation of PKCalpha in a dose-dependent manner, and inhibition or downregulation of PKCalpha blocked cytochalasin D-induced chondrogenesis. Cytochalasin D also downregulated Erk-1 phosphorylation that is associated with chondrogenesis. Our results, therefore, suggest that disruption of actin cytoskeleton induces chondrogenesis of mesenchymal cells by activating PKCalpha and by inhibiting Erk-1 signaling.

Protein kinase C regulates chondrogenesis of mesenchymes via mitogen-activated protein kinase signaling.

A possible regulatory mechanism of protein kinase C (PKC) in the chondrogenesis of chick limb bud mesenchymes has been investigated. Inhibition or down-regulation of PKC resulted in the activation of a mitogen-activated protein kinase subtype Erk-1 and the inhibition of chondrogenesis. On the other hand, inhibition of Erk-1 with PD98059 enhanced chondrogenesis and relieved PKC-induced blockage of chondrogenesis. Erk-1 inhibition, however, did not affect expression and subcellular distribution of PKC isoforms expressed in mesenchymes nor cell proliferation. The results suggest that PKC regulates chondrogenesis by modulating Erk-1 activity. Inhibition or depletion of PKC inhibited proliferation of chondrogenic competent cells, and Erk-1 inhibition did not affect PKC modulation of cell proliferation. However, PKC-induced modulation of expression of cell adhesion molecules involved in precartilage condensation was reversed by the inhibition of Erk-1. Expression of N-cadherin was detected at the early period of chondrogenesis. Inhibition or depletion of PKC induced sustained expression of N-cadherin, and Erk-1 inhibition blocked the effects of PKC modulation. The expression of integrin alpha5 beta1 and fibronectin was found to be increased transiently during chondrogenesis. Depletion or inhibition of PKC caused a continuous increase of the expression of these molecules throughout the culture period, and Erk-1 inhibition abolished the modulating effects of PKC. Because reduction of the examined cell adhesion molecule expression is a prerequisite for the progression of chondrogenesis after cell condensation, our results indicate that PKC regulates chondrogenesis by modulating expression of these molecules via Erk-1 signaling.

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

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.

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.

Expression of protein kinase C isozymes that are required for chondrogenesis of chick limb bud mesenchymal cells.

Protein kinase C (PKC) has been suggested to be involved in the chondrogenesis of chick limb bud mesenchymal cells. This study examined the expression and the role of PKC isozymes in chondrogenesis. Multiple PKC isozymes such as conventional PKC (cPKC alpha and gamma), new PKC (nPKC epsilon), and atypical PKC (aPKC zeta, lambda, and tau) were expressed in chondroblasts but cPKC beta and nPKC delta were not detected. The amounts of expressed cPKC and nPKC isozymes, namely cPKC alpha and gamma and nPKC epsilon, were increased as chondrogenesis proceeds while the level of aPKC isozymes was not changed. Treatment of cells with specific PKC inhibitors blocked chondrogenesis. Prolonged exposure of cells to phorbol ester which down regulates both cPKC and nPKC also blocked chondrogenic differentiation. The inhibition of chondrogenesis was the most effective when PKC activity was blocked at the early stage of chondrogenesis (i.e., for the first 24 hours of micromass culture). Down regulation of PKC blocked both proliferation of cells and synthesis of sulfated proteoglycans, indicating that expression of cPKC and nPKC is required at early stage of chondrogenesis.

Building community for health: lessons from a seven-year-old neighborhood/university partnership.

This article presents two case studies highlighting the role of community conflict in the process of community empowerment. A graduate program for community health nurses (CHNs) in a large Midwestern city formed a partnership with a diverse, integrated neighborhood for the dual purposes of enhancing the community's capacity to improve its own health and teaching CHNs community organizing as a means to improve health. Central to the partnership are a broad definition of health, trust developed through long-term involvement, a commitment to reciprocity, social justice, and Freire's model of adult learning. Two initiatives that gave rise to major conflicts between community groups are analyzed. Conflicts, external and internal to the community, proved to be both powerful catalysts and potential barriers to the use of Freirian themes in community organization. Both university and community participants report needing better skills in the early recognition and management of conflict. We conclude that conflict management theory must be integrated with empowerment education theory, particularly when empowerment education is applied in a diverse community.

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.

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.

Effect of isoproterenol on regional myocardial segment work, O2 consumption, and oxygen balance.

We tested the hypothesis that positive inotropic stimulation by isoproterenol alters the relationship between regional segment work and regional myocardial oxygen consumption. Regional parameters were compared with external cardiac work and global LV oxygen consumption. In anesthetized open-chest dogs, regional myocardial segment length (ultrasonic dimension crystals) and force development (miniature force transducer) were measured. The integrated multiples of myocardial shortening by corresponding force during an averaged beat expressed segment work (area under the systolic portion of the length-force loop). External cardiac work was calculated from aortic blood pressure and cardiac output. Global and regional myocardial MVO2 were evaluated at baseline and during intravenous infusion of isoproterenol (0.5 and 1.0 micrograms/kg per min). Regional coronary blood flow was measured with radioactive microspheres, and microspectrophotometry of frozen myocardial biopsies was used to evaluate O2 saturation in small arteries and veins. These parameters were used to calculate regional MVO2. Arterial and coronary sinus O2 saturation was used to calculate global LV O2 consumption. Regional myocardial O2 balance was estimated by measurement of NADH redox level using surface fluorometry. It was found that 0.5 micrograms/kg per min isoproterenol increased regional segment work/minute from 4650 +/- 495 to 6750 +/- 750 mm.g/min. Corresponding regional oxygen consumption was disproportionately increased from 5.43 +/- 0.61 to 15.24 +/- 1.37 ml/min per 100 g. External cardiac work was found to decrease from 728 +/- 13 to 562 +/- 25 mmHg.1/min (due to decreased aortic blood pressure), whereas global myocardial O2 consumption increased. Regional myocardial O2 extraction and NADH fluorescence were elevated, indicating impaired tissue oxygenation. Regional MVO2 was increased by 153 +/- 56%, but regional work by only 45.3 +/- 33% (P < 0.05). These results indicate that regional contraction efficiency was markedly reduced by isoproterenol.

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.

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)