Avis L. Sylvia

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.

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.

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.

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.

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.

Fluorometric monitoring of the effects of adrenergic agents on oxidative metabolism in intact cerebral cortex

Abstract Amphetamine and other adrenergic agents were shown to alter the rate of oxidative metabolic activity measured by nicotinamide-adenine dinucleotide (NADH) fluorometry in intact cerebral cortex of cats and rats. Both amphetamine and isoproterenol administration produced a similar triphasic metabolic response pattern in brain: (1) an initial decrease in the NADH level, followed by (2) a more prolonged increase in the NADH redox level, and (3) a later large decrease in the level of reduced coenzyme. This pattern is interpreted to reflect increased “recovery” metabolism associated with enhanced cellular activity. Reserpine administration resulted in similar metabolic redox patterns except this drug produced a later increase in NADH level to a greater than baseline level. Chlorpromazine and propranolol increased the NADH level. Propranolol blocked the triphasic response patterns of both amphetamine and isoproterenol. When propranolol or chlorpromazine were given after either amphetamine or isoproterenol, the injection was followed by an increased level of NADH. It is thought that the effects of propranolol, chlorpromazine and reserpine are due to decreased tissue activity while the amphetamine and isoproterenol actions are mediated through central β-receptors. These experiments demonstrate the feasibility of optically monitoring oxidative metabolic activity in situ during drug administration and indicate the importance of combining this technique with biochemical assay procedures.

Abnormalities of Cerebral Oxidative Metabolism with Aging and Their Relation to the Central Noradrenergic System

Cerebral oxidative metabolism was studied in vivo by monitoring redox shifts of cytochrome c oxidase in response to direct electrical stimulation of the cerebral cortex in Fischer-344 rats at 3 and 28 months of age. Such activation results in a transient oxidation of cytochrome oxidase associated with brief increase in local cerebral blood volume. In aged rats, the rates of the transient redox responses of cytochrome oxidase (i.e. initial oxidation followed by re-reduction) are slowed by about 50% in comparison to young rats. Cortical norepinephrine was similar in both age-groups. However, while depletion of cortical norepinephrine causes slowing of the rate of re-reduction in young rats by about 50%, such depletion had no effect on the already slow kinetics of the redox shifts of aged rats. Vascular reactivity to increased metabolic demands, defined by the amplitude ratio of the blood volume increase to the cytochrome oxidation, is increased with age but attenuated by norepinephrine depletion in both age-groups. These results suggest that: (1) cerebral cortical levels of norepinephrine do not decline with age in the Fischer-344 rat; (2) development of an age-related impairment in the capability of cerebral oxidative metabolism to respond to conditions of heightened metabolic demands; (3) such impairment is not worsened by depletion of cerebral norepinephrine; (4) exaggerated vascular reactivity to increased metabolic requirement in aging indicates decreased provision of oxygen and metabolic substrates, and (5) this vascular reactivity is mediated by central noradrenergic mechanisms.

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 <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 <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.

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.

The Near Infrared (Nir) Absorption Band of Cytochrome aa3 in Purified Enzyme, Isolated Mitochondria and in The Intact Brain in Situ

The potential value of monitoring the redox state of cytochrome c oxidase (cytochrome aa 3 or cyt aa 3) as an index to cerebral energetics was highlighted in the preceding contribution (Sylvia et al., 1985), Hypoxically induced changes in the redox state of cyt aa 3 correlated well with creatine phosphate fluctuations. That study was performed using the well described absorption peak of reduced heme a. in the visible part of the spectrum as the marker (Jobsis et al., 1977). Even as successful as that effort has proved to be, a considerable drawback exists in the fact that, except for very thin skulled animals such as rats and birds, the cerebral cortex must be exposed to obtain the optical signals. In order to be maximally useful, a totally non-invasive approach should be provided for observations on larger species including humans. Not only would this provide a greater range of applicability, including routine monitoring of patients, but the importance of preserving normal intra-cranial pressure relations can hardly be overstressed.