Reactivity of the cerebrocortical vasculature and energy metabolism to direct cortical stimulation in haemorrhagic shock.

Direct electrical stimulation of the cerebral cortex was used to determine the changes of cortical carbohydrate and oxidative metabolism and of vascular reactivity during haemorrhagic shock. The results were as follows. 1. Electrical stimulation of the brain cortex applied in the control period led to a marked vasodilation and NAD reduction that was preceded in part of the experiments by a transient NADH oxidation. It is suggested that the increase in cortical NADH fluorescence observed during direct stimulation is due to the fact that the rate of cytoplasmic NADH production exceeded the rate of mitochondrial NADH oxidation and of the rate of H+-transport from the cytoplasm into the mitochondria. 2. The cerebrocortical vascular and NAD/NADH redox state responses induced by electrical stimulation changed in the early hypovolaemic phase of shock. At this time, electrical stimulation of the brain cortex led to NADH oxidation in the majority of the experiments or in some experiments, the stimulation did not bring about changes in the redox state of the cortex. The total loss of the reactivity to direct stimulation of the cerebrocortical vessels and of energy metabolism preceded the occurrence of cortical ischaemia during the hypovolaemic phase of shock. 3. Since after reinfusion of the shed blood, redox state and vasculature remained unresponsive to stimulation even in those experiments in which the cortical ischaemia improved, it is concluded that the carbohydrate and oxidative metabolism of the brain cortex were already irreversibly damaged in the early phase of hypovolaemic shock.     

Effect of arterial hypoxia on the cerebrocortical redox state, vascular volume, oxygen tension, electrical activity and potassium ion concentration.

The effect of different degrees of arterial hypoxia on cerebrocortical NAD/NADH redox state, reflectance, oxygen tension, extracellular potassium ion concentration, ECoG and arterial blood pressure was investigated in rats. The results may be summarized as follows. a) The decrease of cortical pO2 preceded the dilatation of cortical vessels by 15-20 sec but the changes in cortical extracellular potassium ion concentration, ECoG and arterial blood pressure started later than the vasodilatation. These results give further support to the regulatory role of cortical pO2 decrease in the initiation of cerebrocortical vasodilatation during arterial hypoxia. b) Since the K+ concentration of the brain cortex and the ECoG did not change in mild arterial hypoxia, the significant NAD reduction obtained in this experimental group is likely to be of cytoplasmic origin. The same conclusion applies to the initial periods of severe arterial hypoxia. On the basis of the extent of NAD reduction during various degrees of arterial hypoxia it is concluded that about 30% of the NAD reduction occurring in anoxia is of cytoplasmic origin. c) When the animals were ventilated with a gas mixture containing 4-7% oxygen, the brain cortex became nearly anoxic, partly because of the gradual decrease of arterial blood pressure. Finally, the mechanism of potassium leakage is identical under prolonged severe arterial hypoxaemia and on anoxic terminal depolarization.     

Mechanism of the cerebrocortical vasodilatation during anoxia.

The possible role of cerebrocortical ion homeostasis, NAD/NADH redox state and of cortical oxygen tension was investigated in the initiation of hypoxic cortical vasodilatation. In addition, changes in cerebrocortical extracellular concentrations of Na+, K+, and Cl- during anoxia were studied. The results were as follows. a) The cerebrocortical reflectance decrease, e.g. cerebral vasodilatation, lagged behind the cortical pO2 decrease by 1-2 sec, but preceded the decrease of arterial blood pressure and ECoG as well as the extracellular Na+, K+, Cl- increases by 20-30 sec. Since the cortical pO2 decreased first and the ion changes lagged behind the onset of vasodilatation by 20-30 sec, it is suggested that the CBF increase in hypoxia is mediated via the cortical pO2 decrease. b) A significant NAD reduction was already present after 20 sec. of nitrogen breathing. Since the ECoG and MABP decreased, and K+ activity increased much later than this, it is presumed that the NAD reduction during the first 30-40 sec of anoxia indicates an increased rate of glycolysis, but not mitochondrial hypoxia. c) In the predepolarization phase a 17% K+, 4% Na+, 5% Cl- increase is probably the result of a reduction of the extracellular spaces caused by water movement and by the migration of Na+ and Cl- from the extracellular to the intracellular space. The large K+, Na+, Cl- changes during terminal depolarization can be interpreted as a result of the failure of the membrane bound Na+ -K+ pump and of the altered ion permeability of the cell membranes.     

A Simple Cranial Window Technique for Optical Monitoring of Cerebrocortical Microcirculation and NAD/NADH Redox State. Effect of Mitochondrial Electron Transport Inhibitors and Anoxic Anoxia

Abstract: Fluorescence of NADH and vascular volume of the brain cortex of chloralose-anesthetized cats were measured by surface fluororeflectometry. A cranial window and superfusion technique was elaborated for the topical inhibition of mitochondrial electron transport in the brain cortex by amytal (inhibits at site I) and cyanide (inhibits at site III). The changes in NAD/NADH redox state and CVV evoked by these electron transport inhibitors were compared with those elicited by anoxic anoxia. Amytal (10^-3-10^-1 M ) and cyanide (10^-5-10^-2 M ) resulted in a concentration-dependent and reversible increase in cortical NAD reduction and vascular volume, but the cerebrocortical vessels were almost completely dilatated long before maximum NAD reduction was reached. Cyanide at 10^-2 M increased cortical NAD reduction and vascular volume as much as anoxic anoxia. Amytal at 10^-1 M induced approximately half of the NAD reduction evoked by 10^-2 M cyanide or anoxic anoxia, but resulted in only slightly less vasodilatation than that following cyanide and anoxic anoxia. Since amytal inhibits mitochondrial electron transport at site I—and cyanide and anoxia at site III—but induces a comparable degree of vasodilatation, it is concluded that cytochrome oxidase cannot be the single molecular oxygen sensor in the brain cortex.     

Effect of aging on brain energy-metabolism

The aging process involves morphological and functional changes in cerebral vasculature and deterioration of mitochondrial number and function. Furthermore, slow oscillations of cerebral blood flow and oxidative metabolism occur in animals under different pathological conditions such as ischemia. The aim of this study was to evaluate the effect of aging on energy-metabolism of the rat brain during anoxia and normoxia and to further investigate the occurrence of oscillations under normoxia in the aging brain. Simultaneous hemodynamical (CBF), biochemical (NADH/NAD ratio) and electrical activity from the cerebral cortex were measured by means of a multiparametric assembly (MPA) system. Exposure of adult rats to anoxia (100% N(2)) resulted in a 36+/-2% elevation of NADH. Furthermore, exposure of the aged group to anoxia caused NADH elevation as low as 9.6+/-4% (P<0.05). The changes in the NADH levels were followed by an increase in CBF. In addition, during the normoxic periods, hemodynamic oscillations were recorded in the old animals. This study suggests that the structural and functional changes that occur in vessels in the aging brain cause disability of cerebromicrovessels to optimally deliver nutrients and oxygen to the brain, affecting the mitochondrial ability to respond to anoxia. Furthermore, this study supports the approach that the hemodynamic oscillations are related to the development of a pathological state and are not a normal cerebral function.     

Comparison of cerebral NADH and cytochrome aa3 redox shifts during anoxia or hemorrhagic hypotension.

The reduction-oxidation reactions of NADH and cytochrome $\text{aa}{}_3$ to incipient oxygen insufficiency caused by nitrogen ventilation or hemorrhagic hypotension were examined in the exposed cerebral cortex of the cat. A comparison of the onset of redox changes with each procedure shows that cytochrome $\text{aa}{}_3$ reduction precedes the reduction of mitochondrial NAD. This constitutes evidence that, in the living brain, NADH maintains its resting oxidation state at lower cellular oxygen tensions than cytochrome $\text{aa}{}_3$ does, consistent with the differences in oxygen affinity these respiratory chain components exhibit during oxygen titration $\text{in vitro}$.     

Lack of protective effect of adrenaline tolerance haemorrhagic shock in conscious dogs

Dogs were made tolerant to lethal doses of adrenaline by treatment with increasing doses of the hormone up to 1 mg/kg. The conscious animals were then subjected to haemorrhagic shock with a hypovolaemic period of 3 hours. Survival was 8/17 in the pretreated group and 15/18 in the control group. 12 of 29 dogs died already during adrenaline treatment. Plasma catecholamine levels were higher in the treated group already before bleeding and also during hypovolaemia. Catecholamine induced myocardial lesions were found in the treated group. No evidence of a blunting of the sympathetic response or a protective effect in haemorrhagic shock was seen in adrenaline tolerance.     

Effects of acetazolamide on cerebrocortical NADH and blood volume

Acetazolamide (AZ), a potent carbonic anhydrase inhibitor in human and animal tissues, increases cerebral blood flow (CBF) by acidifying cerebral extracellular fluids. To demonstrate the relationship of increased CBF to brain O2 availability after AZ administration, a compensated fluorometer was used to study changes in the cerebrocortical redox balance in rabbits. Seven rabbits were anesthetized with pentobarbital sodium. Excitation light (366 nm) was conducted to the cerebrocortical surface of each animal by a 4-mm-diam fiberoptic light guide. Fluorescence emissions from cerebrocortical NADH (450 nm) were compared at different inspired O2 (FIO2) tensions. Reflected light (366 nm), which was used to determine a correction to the fluorescence signal, was separately quantitated and interpreted as an index of cerebrocortical blood volume. Reductions in FIO2 from 1.0 to 0.21, 0.14, 0.10, and 0.07 resulted in increases in both tissue blood volume and [NADH]. Intravenous AZ (25 mg/kg) increased cerebrocortical blood volume and reduced the [NADH], even during ventilation with 100% O2. The changes in brain redox balance caused by vasodilation with AZ were compared with those caused by vasodilatation with CO2. The NAD+/NADH redox state was a continuous function of FIO2 at all levels of arterial PCO2 (PaCO2), both before and after AZ administration. The improvement in cerebral O2 delivery caused by AZ-induced vasodilation was comparable to that caused by the vasodilatation that results from a PaCO2 elevation approximately equal to 12-15 Torr above normal. The slope of the relationship between [NADH] and FIO2 was similar at normal, low, and high levels of PaCO2. We conclude that AZ administration and PaCO2 elevation improve cerebral oxygenation by similar mechanisms.     

A relation between (NAD+)/(NADH) potentials and glucose utilization in rat brain slices.

Changes in several parameters involved in the control of metabolism were correlated with changes in glucose utilization in rat brain slices incubated under conditions which reduced glucose oxidation by 40 to 70%. The parameters included: the concentrations of ATP, ADP, AMP, and the adenylate energy charge; the cytoplasmic oxidation-reduction state ([NAD+]/[NADH]), determined from the [pyruvate]/[lactate] equilibrium; the mitochondrial oxidation-reduction state, determined from the [NH4+] ]2-oxoglutarate]/[glutamate] Equilibrium; the cytoplasmic and mitochondrial oxidation-reduction potentials (in volts), calculated from the respective [NAD+]/ [NADH] ratios using the Nernst equation; and the difference between the cytoplasmic and mitochondrial [NAD+]/[NADH] potentials. The conversion of [3, 4-14C] glucose to 14CO2 and of [U-14C] glucose to acetylcholine and to lipids, proteins, and nucleic acids by the brain slices were also determined. The values obtained by subtracting the mitochondrial from the cytoplasmic [NAD+1/[NADH] potentials correlated more closely with glucose utilization than did other parameters, under the conditions studied. For the synthesis of acetylcholine, the correlation coefficient was 0.96, and for the production of 14CO2 from [3, 4-14C] glucose it was 0.82.     

Studies on the effects of coenzyme A-SH: acetyl coenzyme A, nicotinamide adenine dinucleotide: reduced nicotinamide adenine dinucleotide, and adenosine diphosphate: adenosine triphosphate ratios on the interconversion of active and inactive pyruvate dehydrogenase in isolated rat heart mitochondria.

The content of coenzyme A-SH (CoASH) and acetyl-CoA of suspensions of rat heart mitochondria was stabilized by the addition of DL-carnitine and acetyl-DL-carnitine, in the presence of the respiratory inhibitor rotenone. The mitochondrial content of NAD+ and NADH was similarly stabilized by the addition of acetoacetate and DL-3-hydroxybutyrate, and the content of ADP and ATP was imposed by the addition of these nucleotides to the mitochondrial suspension, in the presence of uncoupling agent and oligomycin, to inhibit ATPase. Under these conditions, mitochondrial CoASH/acetyl-CoA, NAD+/ NADH, and ADP/ATP ratios could be varied independently, and the effect on the interconversion of active and inactive pyruvate dehydrogenase could be studied. Decreases in both CoASH/acetyl-CoA and NAD+/NADH ratios were shown to be inhibitory to the steady state activity of pyruvate dehydrogenase, and this effect is described at three different ADP/ATP ratios and different concentrations of added MgCl2. A new steady state level of activity was achieved within 10 min of a change in either CoASH/acetyl-CoA or NAD+/NADH ratio; the rate of inactivation was much higher than the rate of reactivation under these conditions. Effects of CoASH/acetyl-CoA and NAD+/NADH may be additive but are still quantitatively lesser than the changes in activity of pyruvate dehydrogenase induced by changes in ADP/ATP ratio. The variation in activity of pyruvate dehydrogenase with ADP/ATP ratio is described in the absence of changes in the other two ratios, conditions which were not met in earlier studies which employed the oxidation of different substrates to generate changes in all three ratios.     

Role of NADH/NAD+ transport activity and glycogen store on skeletal muscle energy metabolism during exercise: in silico studies

Skeletal muscle can maintain ATP concentration constant during the transition from rest to exercise, whereas metabolic reaction rates may increase substantially. Among the key regulatory factors of skeletal muscle energy metabolism during exercise, the dynamics of cytosolic and mitochondrial NADH and NAD+ have not been characterized. To quantify these regulatory factors, we have developed a physiologically based computational model of skeletal muscle energy metabolism. This model integrates transport and reaction fluxes in distinct capillary, cytosolic, and mitochondrial domains and investigates the roles of mitochondrial NADH/NAD+ transport (shuttling) activity and muscle glycogen concentration (stores) during moderate intensity exercise (60% maximal O2 consumption). The underlying hypothesis is that the cytosolic redox state (NADH/NAD+) is much more sensitive to a metabolic disturbance in contracting skeletal muscle than the mitochondrial redox state. This hypothesis was tested by simulating the dynamic metabolic responses of skeletal muscle to exercise while altering the transport rate of reducing equivalents (NADH and NAD+) between cytosol and mitochondria and muscle glycogen stores. Simulations with optimal parameter estimates showed good agreement with the available experimental data from muscle biopsies in human subjects. Compared with these simulations, a 20% increase (or approximately 20% decrease) in mitochondrial NADH/NAD+ shuttling activity led to an approximately 70% decrease (or approximately 3-fold increase) in cytosolic redox state and an approximately 35% decrease (or approximately 25% increase) in muscle lactate level. Doubling (or halving) muscle glycogen concentration resulted in an approximately 50% increase (or approximately 35% decrease) in cytosolic redox state and an approximately 30% increase (or approximately 25% decrease) in muscle lactate concentration. In both cases, changes in mitochondrial redox state were minimal. In conclusion, the model simulations of exercise response are consistent with the hypothesis that mitochondrial NADH/NAD+ shuttling activity and muscle glycogen stores affect primarily the cytosolic redox state. Furthermore, muscle lactate production is regulated primarily by the cytosolic redox state.     

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.     

Temperature Coefficients for the Oxidative Metabolic Responses to Electrical Stimulation in Cerebral Cortex

Temperature coefficients of both cat and toad brain have been calculated for the active metabolic state induced by electrical stimulation. Values are higher than most of the values previously reported for "rest" metabolism, whether calculated from Arrhenius plots or from linear graphs. Relative rates of oxidative metabolism were obtained by measuring the time course of the transient changes in NADH fluorescence and cytochrome aa3 absorption by reflectance techniques directly from the surface of the exposed cat cerebral cortex in vivo and from the isolated intact toad brain mounted in a cuvet. These findings demonstrate that such optical methods accurately record metabolic processes.     

Nitrogen metabolism in the isolated perfused rat liver. Nitrogen balance, redox state and rates of proteolysis

Nitrogen balances were measured in isolated perfused rat livers in the presence and absence of nitrogen donors. In all instances the balance apparently was incomplete. The expression [alanine][alpha-oxoglutarate]/[pyruvate][glutamate] remained fairly constant under the metabolic conditions studied, indicating that it may be at near-equilibrium. The source of the extra nitrogen seems to be derived from increased hepatic proteolysis. The addition of a nitrogen donor to the perfusate arrested proteolysis, as did the addition of pyruvate. The free mitochondrial [NAD(+)]/[NADH] ratio, calculated from the glutamate dehydrogenase and beta-hydroxybutyrate dehydrogenase reactants, showed similar values and exhibited parallel changes under most metabolic situations studied. These results suggest that, under the reported experimental conditions, both dehydrogenases share a common mitochondrial NAD pool. Glutamate dehydrogenase plays an important role in hepatic nitrogen metabolism in vivo.     

Prooxidant activity of fisetin: effects on energy metabolism in the rat liver

Flavonols, which possess the B-catechol ring, as quercetin, are capable of producing o-hemiquinones and to oxidize NADH in a variety of mammalian cells. The purpose of this study was to investigate whether fisetin affects the liver energy metabolism and the mitochondrial NADH to NAD+ ratio. The action of fisetin on hepatic energy metabolism was investigated in the perfused rat liver and isolated mitochondria. In isolated mitochondria, fisetin decreased the respiratory control and ADP/O ratios with the substrates α-ketoglutarate and succinate. In the presence of ADP, respiration of isolated mitochondria was inhibited with both substrates, indicating an inhibitory action on the ATP-synthase. The stimulation of the ATPase activity of coupled mitochondria and the inhibition of NADH-oxidase activity pointed toward a possible uncoupling action and the interference of fisetin with mitochondrial energy transduction mechanisms. In livers from fasted rats, fisetin inhibited ketogenesis from endogenous sources. The β-hydroxybutyrate/ acetoacetate ratio, which reflects the mitochondrial NADH/NAD+ redox ratio, was also decreased. In addition, fisetin (200 μM) increased the production of (14)CO2 from exogenous oleate. The results of this investigation suggest that fisetin causes a shift in the mitochondrial redox potential toward a more oxidized state with a clear predominance of its prooxidant activity.     

Use of noninvasive fluorometry and spectrophotometry to study epithelial metabolism and transport

Various examples illustrating the use of spectrophotometry and fluorometry in epithelia are presented. The first example uses the redox level of cytochrome aa3, measured spectrophotometrically as an index of tissue anoxia in cortical tubules and slices from the rabbit kidney. In the second example the redox level is used to measure the kinetics of aerobic energy production during transition to anoxia in the midgut of the tobacco hornworm. In the third application, the redox level of mitochondrial NADH is measured fluorometrically in a cortical tubule suspension from the rabbit kidney. Inhibition of active transport work causes reduction of NAD whereas increased work elicits oxidation of NAD, both occurring as expected from mitochondrial transitions to a lesser or more active state, respectively. Another use of NADH fluorescence is the determination of the relative effectiveness of metabolic substrates to deliver reducing equivalents to the respiratory chain in a particular tissue. Redox changes in mitochondrial NAD may be used to distinguish between primary metabolic and primary transport effects of hormones, drugs, and changes in the state of the organism. Finally, examples are provided of the use of an intracellular pH-sensitive dye and an extracellular calcium-sensitive dye in kidney tubules.     

Effect of hypothermia on brain multi-parametric activities in normoxic and partially ischemic rats

Hypothermia, as well as anesthesia, are known to protect the brain against ischemia, hypoxia and other pathological damages. One of the mechanisms of this improvement could be by lowering brain function, and thereby lowering oxygen demand. We examined the effect of hypothermia on brain function and blood supply in awake and anesthetized rats and studied the interaction between partial ischemia and the responses to hypothermia. The brain function multiprobe (BFM) used enabled simultaneous measurements of cerebral blood flow (CBF), mitochondrial NADH redox state, extracellular K(+) concentration, DC potential and ECoG from the cerebral cortex in rats whose brain temperature was lowered by 5 degrees C. Hypothermia was induced in awake, anesthetized and brain ischemic-anesthetized rats. In anesthetized and ischemic-anesthetized rats, the time required for lowering the brain temperature by 5 degrees C was five times less than in the normal awake animals. No significant changes in CBF and NADH levels were found in response to hypothermia in the awake animals. In contrast, a significant decrease in extracellular K(+) concentration was recorded under hypothermia, probably due to the lower rate of depolarization. Hypothermia in anesthetized and in ischemic-anesthetized rats did not significantly affect the levels of mitochondrial NADH, CBF and extracellular K(+). Hypothermia under ischemia was expected to be more effective.     

Aminooxyacetic acid inhibits the malate-aspartate shuttle in isolated nerve terminals and prevents the mitochondria from utilizing glycolytic substrates

Aminooxyacetate, an inhibitor of pyridoxal-dependent enzymes, is routinely used to inhibit gamma-aminobutyrate metabolism. The bioenergetic effects of the inhibitor on guinea-pig cerebral cortical synaptosomes are investigated. It prevents the reoxidation of cytosolic NADH by the mitochondria by inhibiting the malate-aspartate shuttle, causing a 26 mV negative shift in the cytosolic NAD+/NADH redox potential, an increase in the lactate/pyruvate ratio and an inhibition of the ability of the mitochondria to utilize glycolytic pyruvate. The 3-hydroxybutyrate/acetoacetate ratio decreased significantly, indicating oxidation of the mitochondrial NAD+/NADH couple. The results are consistent with a predominant role of the malate-aspartate shuttle in the reoxidation of cytosolic NADH in isolated nerve terminals. Aminooxyacetate limits respiratory capacity and lowers mitochondrial membrane potential and synaptosomal ATP/ADP ratios to an extent similar to glucose deprivation. Thus, the inhibitor induces a functional 'hypoglycaemia' in nerve terminals and should be used with caution.     

Effect of surplus amount of oxygen on the cerebrocortical microcirculatory reactions associated to moderate arterial hypotension

The aim of the present study was to clarify whether tissue hypoxia is involved in the autoregulatory dilatation of cerebrocortical vessels occurring at moderate arterial hypotension. In order to avoid hypoxia that may occur during arterial hypotension, in one part of the experiments the brain cortices were superfused with oxygen saturated (pO2, approximately 500 mm Hg) artificial cerebrospinal fluid (mock CSF). In the other part of the experiments arterial hypotension was induced without superfusing the brain cortices (closed skull). Mean arterial blood pressure (MABP) was decreased in both experimental groups by bleeding to 75-85 mm Hg for approximately 5 min, then the shed blood was reinfused. Changes in cortical vascular volume (CVV), mean transit time of cortical blood flow (tm), and blood flow (CBF) were measured through a cranial window with a microscope reflectometer. Although CSF pO2 differed markedly between the superfused and nonsuperfused experimental groups, arterial hypotension led to similar changes in CVV and tm in both groups. Due to the proper dilatation of the cerebrocortical arterioles, CBF was not altered by arterial hypotension in either of the groups. These results suggest that the brain cortex does not become hypoxic at moderate arterial hypotension and, consequently, incipient tissue hypoxia has no role in the autoregulatory dilatation of the cerebrocortical arterial network.