Cyanide-induced cytochrome a,a3 oxidation-reduction responses in rat brain in vivo.

The sensitivity of the brain to cyanide-induced histotoxic hypoxia and the protective effects of known cyanide antagonists, have been assessed in vivo by reflectance spectrophotometry. Cyanide-related changes in cytochrome a,a3 (cytochrome c oxidase) oxidation-reduction (redox) state, tissue hemoglobin saturation, and local blood volume were continuously monitored in cerebral cortex of rats. Noncumulative, dose-dependent inhibition of the in situ mitochondrial respiratory chain was evaluated directly by measuring increases in reduction levels of the terminal oxidase. These transient cytochrome a,a3 reductions were accompanied by increases in regional cerebral hemoglobin saturation and blood volume. Cytochrome redox responses were not altered either in magnitude or kinetics by hyperoxia; however, the cyanide-cytochrome dose-response curve was greatly shifted to the right by pretreatment with sodium nitrite, and the recovery rate of cytochrome a,a3 from cyanide-induced reduction was enhanced fourfold by pretreatment with sodium thiosulfate.

O2 dependence of in vivo brain cytochrome redox responses and energy metabolism in bloodless rats.

Oxygen-dependent changes in brain cytochrome redox state and cerebrocortical energy metabolism were evaluated in fluorocarbon-circulated rats at hematocrits of less than 1%. Redox levels of three respiratory chain cytochrome complexes, b, c, and a,a3 (cytochrome c oxidase), were continuously measured directly through the intact skulls of animals using reflectance spectrophotometry. The in vivo redox status of cytochromes at different FiO2 was directly compared with in vitro measured changes in cortical metabolites known to reflect energy production, i.e., glucose, pyruvate, lactate, phosphocreatine (PCr), ADP, and ATP. Lowering the FiO2 to less than 1.0 caused the cytochromes to become increasingly more reduced. This was associated with increased tissue accumulation of pyruvate and lactate and a concomitant increase in the lactate/pyruvate (L/P) ratio. At FiO2 = 0.6, cytochromes b, c, and a,a3 were 57, 53, and 46% reduced, respectively. There was no apparent cerebral energy deficit since changes in cortical PCr, ADP, and ATP concentrations were not statistically significant. Bloodless animals did not survive below FiO2 = 0.5. At this FiO2, the inability of the animals to sustain arterial pressure correlated (r = 0.87) with depletion of PCr and further increases in the L/P ratio (r = 0.66). Yet, the cortical ATP content was reduced by only 9% of control value. These data provide direct evidence that fluorocarbon emulsion (FC-43) sustains brain oxygenation and energy metabolism at high partial pressures of molecular O2. At lower FiO2, however, mitochondrial O2 uptake becomes limited as a function of decreasing perfusion pressure.

Direct effects of CO on cerebral energy metabolism in bloodless rats.

Cerebrocortical b-cytochromes have been found to be sensitive to reduction in the presence of CO and O2 in vivo. CO-mediated cytochrome b reduction responses in "bloodless" rats were correlated in this study with changes in concentrations of high energy and glycolytic intermediates measured in cortex after rapid brain freezing. Cytochrome redox state and metabolite concentrations also were compared with cerebral blood flow (CBF) and cerebral metabolic rate for O2 (CMRo2) measured before and after CO administration. No definite biochemical evidence of energy limitation was found in parietal cortex after the fluorocarbon-for-blood exchange; however, CO had direct effects on brain metabolite concentrations. Fifteen-minute CO exposures at inspired CO/O2 of 0.003-0.06 increased cerebrocortical phosphocreatine and ADP and decreased creatine concentration. CO exposure produced no significant changes in either ATP concentration or CMRo2, although CBF increased slightly. These findings may be interpreted to indicate that CO binding to cytochrome aa3 at low CO/O2 in vivo increases extramitochondrial pH relative to that within the mitochondrial matrix. In the process, cytochrome b reduction levels increase, possibly signaling an increased efficiency of oxidative phosphorylation relative to O2 uptake by unblocked respiratory chains.

Differences in brain cytochrome responses to carbon monoxide and cyanide in vivo.

Cytochrome oxidation-reduction responses to two mitochondrial electron transport inhibitors, carbon monoxide (CO) and cyanide (CN), were studied in the intact brains of fluorocarbon-circulated rats. In vivo reflectance spectrophotometry indicated that cortical b-type cytochromes (564 nm) were highly resistant to reduction by CN in the presence of O2 but showed reduction responses to the administration of 1-5% CO in 90% O2. In contrast, cyanide-sensitive cytochromes aa3 (605 nm) and c + c1 (551 nm) did not increase their reduction levels during exposure to 5% CO in 90% O2. The in vivo CO-mediated b-cytochrome reduction responses did not occur after pretreatment with the cytochrome b inhibitor, antimycin A. Transmission spectrophotometry of superfused hemoglobin-free rat brain slices confirmed cortical b-type cytochromes to be CN-resistant in the presence of O2. Another cytochrome absorbing at 445 nm also was resistant to reduction by 1-mM cyanide in vitro, but it could be reduced anaerobically. The reduced 445-nm cytochrome bound CO in the presence of cyanide. We postulate that this CN-resistant CO binding component might account for in vivo cytochrome aa3-CO interactions and directly or indirectly modulate cytochrome b reduction responses to CO. In any event, the spectral data indicate different primary tissue target sites for CO and CN in living rat brain and also suggest different bioenergetic consequences of exposure to the two agents.

Carbon monoxide-cytochrome interactions in the brain of the fluorocarbon-perfused rat.

Reflectance spectrophotometry through the skull was used to investigate carbon monoxide (CO) binding by tissue hemoproteins in the brains of barbiturate-anesthetized Sprague-Dawley rats. After splenectomy and extensive perfluorotributylamine exchange transfusion, steady-state spectral scans were obtained in Soret and visible wave-length regions during O2 ventilation, during subsequent exposure to O2-enriched gases containing 1, 3, or 5% CO, and finally after N2 anoxia. These CO exposures were well-tolerated and electroencephalograph (EEG) activity continued to be present. Initial difference spectra were influenced by CO binding to residual hemoglobin, but spectral evidence of CO-mediated b-type cytochrome reduction was obtained in the visible region as CO concentration was increased to 3 or 5%. This was associated with Soret spectra compatible with formation of the reduced cytochrome a3-CO complex. Reduction of cytochrome a at 605 nm and cytochrome c + c1 at 550 nm was absent. These findings may indicate respiratory chain branching through b cytochromes, either to a separate a3-like oxidase independent of the classical cytochrome aa3 or to an unidentified alternative CO-sensitive oxidase.

Effects of age on brain oxidative metabolism in vivo.

Non-invasive optical techniques were used to monitor the effects of increasing cerebral energy demand on metabolic capabilities and vascular reactivity in young and aged brain. Low level of electrical stimulation of the cortex, in both young (4--7 months) and aged (24--28 months) rat brain, were accompanied by transient oxidations of NADH and cytochrome oxidase (a,a3) as measured by microfluorometry and reflection spectrophotometry respectively. Stimulation sufficient to produce spreading cortical depression was accompanied by an oxidation of both NADH and cytochrome a,a3 in young brain together with an increase in local blood volume. There was either no change or a slight disoxygenation of hemoglobin. In aged brain, however, spreading depression was associated with an oxidation of NADH and a reduction of cytochrome a,a3 together with an increase in local blood volume and an oxygenation of hemoglobin. The present results indicate that the relationship between microcirculation and the terminal oxidase step of the respiratory chain is altered in aged brain when energy demand is high.

The effect of age and lung pathology on cytochrome a,a3 redox levels in rat cerebral cortex.

The steady-state reduction/oxidation (redox) ratio of cytochrome a,a3 in the non-stressed or 'resting' cerebral cortex was compared in 'healthy' mature and aged rats and in animals with varying degrees of lung pathology present. By using noninvasive dual wavelength spectrophotometry, cytochrome a,a3 was found to be approximately 30% reduced under 'resting' conditions in both mature and 'aged' brain. Although no significant age-related or strain differences were apparent, the 'resting' redox level of cytochrome a,a3 was markedly affected by the presence of lung pathology. The redox ratio was lower in animals where lung lesion involvement was not extensive, and higher (indicative or cellular hypoxia) in animals having both extensive acute and chronic lung pathology. These studies demonstate that, regardless of age or lung pathology, the cortical cytochrome a,a3 redox state is labile to changes in the amount of inspired oxygen, a condition differing from that of isolated mitochondria. These results indicate that dysfunction of the mitochondrial respiratory chain is not a direct or primary consequence of chronlogical aging in the 'resting' brain.

In vivo modulation of norepinephrine-induced cerebral oxygenation states by hypoxia and hyperoxia.

The effect of intravenous norepinephrine (NE) administration on three O2-dependent parameters of cerebral oxygenation was studied in the parietal cortex of skull intact anesthetized rats. Reflectance spectrophotometry was used to measure in vivo changes in cortical hemoglobin saturation (Hb/HbO2), blood volume (BV), and cytochrome c oxidase (cyt. a,a3) oxidation-reduction state. The influence of arterial pressure of oxygen (paO2) on norepinephrine-induced changes in cortical microcirculatory O2 delivery and cyt. a,a3 redox state was tested under conditions of normoxia, hypoxia, and hyperoxia. Norepinephrine produced cyt. a,a3 redox changes which were independent of compensatory alterations in cortical blood volume and changes in systemic blood pressure at the tested physiological extremes. During normoxia, NE caused dose-dependent systemic pressure-related increases in the oxidation level of cyt. a,a3. Conversely, in hypoxia NE caused a reduction. Microcirculatory and cyt. a,a3 redox responses to low doses of NE during hyperoxia were similar to those obtained at high doses during normoxia. The kinetic pattern of changes in hemoglobin saturation, cyt. a,a3 redox state, and cortical blood volume during normoxia and hypoxia was consistent with direct alteration in oxygen delivery to the respiratory chain and possible modification of cerebral oxidative metabolism. Blood-brain barrier alterations and vascular smooth muscle resistance changes to NE under tested conditions of oxygenation are postulated to be responsible for the observed results.

Cerebral cytochrome a,a3 inhibition by cyanide in bloodless rats.

Brain cytochrome a,a3 inhibition is presumed to be the site of lethal histotoxic hypoxia in cyanide poisoning perhaps because of the relative inability of the brain to metabolize cyanide. However, only limited data are available about cyanide toxic effects and possible antagonism in the in vivo brain. In this study, in situ, multiple wavelength, spectrophotometric monitoring of brain cytochrome a,a3 was used to observe oxidation-reduction (redox) responses of cerebral cytochrome a,a3 to intravenous potassium cyanide administration. Bloodless rats prepared by perfluorochemical emulsion (FC-43) exchange transfusion allowed monitoring of cyanide-cytochrome a,a3 interaction without spectral interference by hemoglobin. We found that cyanide-induced transient increases in cytochrome a,a3 reduction level and subsequent redox recovery kinetics were similar in bloodless and normal blood circulated rats. Electroencephalographic activity was maintained until a 50% increase in the reduction level of cytochrome a,a3 was induced with cyanide. Pre-treatment with the cyanide antagonist sodium thiosulfate also protected brain cytochrome a,a3 from cyanide-mediated redox state changes by approximately 4-fold both in normal blood circulated controls and during FC-43 circulation. These latter results indicate that sodium thiosulfate, presumably acting at tissue sites of rhodanese activity, can prevent cerebral cytochrome a,a3 reduction by cyanide even in the virtual absence of blood or circulating proteins.

Energy metabolism and in vivo cytochrome c oxidase redox relationships in hypoxic rat brain.

Rats were subjected to graded arterial hypoxia while we measured changes in the oxidation level of cytochrome c oxidase (cytochrome aa3) in the brain by a non-invasive, optical technique. The experiments were terminated at different arterial oxygen tensions (hypoxic levels) and the in vivo observations were compared with in vitro measured changes in metabolites known to reflect limitations in cellular aerobic energy production, e.g. glucose, pyruvate, lactate, phosphocreatine, ATP and ADP. Using absorption changes at 605 nm, in vivo cytochrome aa3 was 46% reduced in normoxia as determined by the range between the maximal oxidation level attained with animals breathing 85% O2 + 15% CO2 and maximal reduction with anoxia (100% N2). Hypoxia reduced cytochrome aa3 to levels of 52, 67, 76, and 84% at mean PaO2 values of 53, 39, 35 and 28 mm Hg, respectively. These increases in reduced cytochrome correlated significantly (r = 0.94) with cortical phosphocreatine depletion, lactate production, and increases in the lactate/pyruvate ratio. However, there were no significant changes in ATP or ADP. Rats did not survive below an FIO2 of 7% because of a precipitous fall in arterial blood pressure. Hypoxically-induced cerebral isoelectricity was coincident with a 50% increase in the cytochrome reduction level (to 73% of the total range defined above). Our results indicate that in vivo monitoring of the reduction level of cytochrome aa3 provides an early, continuous, and direct measure of intracellular oxygen insufficiency at levels which adversely affect aerobic energy production.

Near-infrared monitoring of cerebral oxygen sufficiency. I. Spectra of cytochrome c oxidase.

Near-infrared (NIR) difference spectra were obtained for oxidized cytochrome c oxidase of isolated mitochondria in vitro and of cerebral tissue in situ observed through scalp and skull. The broad peaks of maximal absorption observed in both were not inconsistent with the customary assignment of an 830 nm peak. However, the ratios of the intensity of the NIR band to that of the visible peak (605 nm), which we found to be identical for in-vitro and in-situ spectra, were consistently and significantly higher than those of the various purified enzyme preparations reported in the literature. In addition the half-band widths of our in-vitro and in-situ preparations were narrower. Haemoglobin spectra in the NIR obtained in clear and in highly light-scattering media showed almost total absence of band distortion in this spectral region, suggesting that the differences observed are not due to scattering effects. Anoxia and the specific oxidase inhibitors, cyanide and carbon monoxide, caused the expected disappearance of the band in both the mitochondria in vitro and the cerebrum in situ. The 830 nm band observed in intact, well-oxygenated animal preparations was therefore identified with the NIR absorption band of oxidized cytochrome c oxidase, notwithstanding the differences with the observations on purified preparations. This points to the possibility of developing instrumentation and techniques for the non-invasive monitoring of the redox state of cytochrome c oxidase as an index to cerebral oxygen sufficiency, i.e. adequate delivery and utilization of oxygen to and by brain tissue.

Regional cerebral blood flow in normal blood circulated and perfluorocarbon transfused rats.

Perfluorocarbon blood substitutes have been shown to exert a protective effect in animal models of cerebral ischemia. The mechanisms by which PFCs improve cerebral hemodynamics are uncertain, however decreased viscosity, small particle size and high oxygen solubility relative to plasma are important factors. Extensive perfluorocarbon exchange transfusion (FC-43) in the rat to a hematocrit of 1%, produces a 100% increase in total cerebral blood flow (FIO2 = 1.0, CaO2 = 6 vol%). Similar increases were seen in normal blood circulated animals breathing 12% O2 (CaO2 = 12 vol%). Therefore, immediately following PFC exchange and the resulting decrease in CaO2, oxygen delivery to the brain is maintained by increasing total blood flow in a manner similar to hypoxic hypoxia.

Spectrophotometric monitoring of O2 delivery to the exposed rat kidney.

Optical spectrophotometry was used to measure changes in the oxidation-reduction state of cytochrome c oxidase in the in situ rat kidney. Alterations in the redox state of cytochrome c oxidase were monitored during variations in the amount of oxygen being delivered to the animal. Spectral analyses were performed on whole kidney, cortical tubule suspensions, and blood (the latter flowing freely through surgically implanted femoral arteriovenous shunts). Reaction spectra identified the location of the absorption maxima for reduced cytochrome c oxidase to be at approx. 605 nm. Additional spectral analysis indicated that hemoglobin oxygenation-deoxygenation changes had minimal artifactual interference on the cytochrome redox signals. The present results indicate that mitochondrial cytochrome c oxidase is not maximally oxidized in the in situ kidney of anesthetized rats at arterial oxygen in the in situ kidney of anesthetized rats at arterial oxygen tensions within the normal physiological range. The redox state of the enzyme in vivo is altered by changes in the level of inspired oxygen over a wide range. The current data are consistent with the existence of nonuniform regions of aerobic respiration occurring in the in situ kidney.

Effect of aging on the response of the cerebral cortex to noradrenergic denervation.

Cyclic AMP generation was measured in isolated minces of the ipsilateral and contralateral cortex of young (3-4 months) and aged (28-29 months) Fischer-344 rats, 2 weeks after unilateral lesion of the nucleus locus ceruleus (LC). Measurements were conducted under basal conditions and in the presence of 0.1 mM isoproterenol. Despite marked norepinephrine (NE) depletion of about 90% in the ipsilateral cerebral cortex in both age-groups, cyclic AMP generation under basal conditions was unaffected. However, whereas isoproterenol-stimulated cyclic AMP generation was significantly higher in the NE-depleted ipsilateral cerebral cortex of young rats, such 'denervation supersensitivity' was not apparent in aged rats. Also, endogenous levels of NE were 31% higher, and isoproterenol-stimulated cyclic AMP generation was 33% lower in the contralateral 'control' cortex of aged rats than in young rats. These results suggest an age-related defect in the postsynaptic expression of noradrenergic mechanisms in the rat cerebral cortex. Further research using intermediate ages is needed to establish whether these age-related findings are caused by 'senescence' or are 'developmental' phenomena.

Effect of fluorocarbon-for-blood exchange on regional cerebral blood flow in rats.

Cerebrocirculatory responses to total perfluorocarbon (FC-43)-for-blood replacement (Hct less than 1%) were studied in anesthetized, ventilated rats breathing 100% O2. Changes in total and regional cerebral blood flow (CBF) were measured using the radiolabeled-microsphere technique. The data were compared with two control groups of hemoglobin-circulated animals; one group was exposed to arterial hypoxia (arterial PO2 = 40 Torr) and the other to isovolemic hemodilution with Krebs-Henseleit-albumin (KHA) solution (mean Hct = 11%). Exchange transfusion with FC-43 doubled total and regional CBF, causing preferential flow increases to the cortex and cerebellum. Estimated cerebrovascular resistance fell to 50% of the preexchange value. Both hemodilution and hypoxia control experiments produced CBF responses similar to those obtained in FC-43 animals. Although calculated arterial O2 contents in all three groups of animals were similar, blood viscosity was normal in hypoxic rats and reduced in KHA and FC-43 animals. Since arterial and cerebrovenous PO2s were both high in fluorocarbon-circulated rats, our results suggest that decreased O2 content and perhaps lower viscosity of the circulating fluorocarbon were responsible for increases in CBF required to maintain sufficient delivery of O2 to the brain.

Cyanide-related changes in cerebral O2 delivery and metabolism in fluorocarbon-circulated rats.

Cyanide-induced cytotoxicity is primarily a result of inhibition of O2 uptake by the terminal enzyme of the mitochondrial respiratory chain, cytochrome-c oxidase (cytochrome aa3). The oxidase in the brain is highly vulnerable to cyanide cytotoxicity, but few studies have evaluated the effects of cyanide on cerebral oxygen metabolism. In the present study, we measured oxidation-reduction responses of cerebrocortical cytochrome aa3 to cyanide and related changes in cerebral blood flow (CBF) and O2 metabolism (CMRO2). Accurate measurement of cytochrome aa3 redox state in vivo by reflectance spectrophotometry was accomplished by using fluorocarbon-circulated rats to eliminate spectral interference from hemoglobin. The data indicate that constant intravenous infusions of cyanide caused rapid, progressive reduction responses by cerebrocortical cytochrome aa3 concomitant with increases in CBF of up to 200%. However, CMRO2 was maintained near normal until cerebral O2 delivery began to fall. These cerebral oxidative responses to cyanide may be explained either by redistribution of intracellular O2 supply to mitochondria respiring in an O2-dependent manner or by branching effects within brain mitochondria in vivo.