A3 Redox State Research Articles

Near-infrared and nuclear magnetic resonance spectroscopic assessment of tissue energetics in an isolated, perfused canine hind limb model of dysoxia.

This controlled laboratory study examined the efficacy of near-infrared spectroscopy (NIRS) and 31P-nuclear magnetic resonance (NMR) spectroscopy in measuring regional tissue oxygenation in a isolated, perfused hind limb model of tissue dysoxia. Isolated hind limb perfusion was carried out in 20 mongrel dogs and oxygen delivery was varied by manipulating either hemoglobin concentration, oxygen saturation, or flow. Hind limbs from anesthetized mongrel dogs (n = 20) were separated and isolated perfusion performed. NIRS probes for recording relative O2 saturation of tissue hemoglobin (HbO2) and cytochrome a,a3 and NMR probes for measuring 31P-high energy phosphates were placed over the limb. Measurements of physiologic parameters, blood gases, lactate, NIRS values for HbO2 and cytochrome a,a3 redox state, and 31P-phosphate levels were recorded at set intervals throughout the experiment. Measures of tissue oxygen consumption (VO2) correlated with tissue oxygenation as measured by HbO2 and cytochrome a,a3 redox state (NIRS), as well as by 31P-high energy phosphate levels (NMR) throughout the experiment. Delivery-dependent tissue oxygenation was detected at a higher DO2 by NIRS than by VO2 or NMR. Tissue oxygenation as measured by NIRS and NMR shows excellent correlation with oxygen delivery in an isolated, perfused model of shock. NIRS may allow early detection of tissue dysoxia using rapid non-invasive techniques.

Near-infrared spectroscopy measurement of regional tissue oxyhemoglobin saturation during hemorrhagic shock.

Adequate resuscitation of patients from shock states depends on restoration of oxygen delivery (DO2) to tissues. Direct measurement of systemic DO2 during shock states requires invasive techniques such as pulmonary artery catheterization. These experiments were performed to examine the ability of near-infrared spectroscopy (NIRS), to measure regional tissue oxygenation in a large-animal model of hemorrhagic shock, and to compare these measures to global measures of oxygen delivery. Splenectomized female pigs (n = 11) were anesthetized, instrumented, and monitored. NIRS probes were placed on the leg, in the stomach via nasogastric tube, and on the liver during laparotomy. Hemorrhagic shock was induced by phlebotomy of 28% of blood volume. After 1 hour, resuscitation was with shed blood and crystalloid until cardiac output plateaued. Measurements of physiologic parameters, blood gases, lactate, intramucosal pH, and NIRS values for regional tissue hemoglobin oxygen saturation (StO2), and cytochrome a,a3 redox state were recorded at intervals throughout the experiment. Tissue oxygenation as measured by oxyhemoglobin saturation and cytochrome a,a3 redox (NIRS) correlated with measures of systemic DO2 throughout the experiment. The liver probe demonstrated blunted changes in tissue oxygenation suggesting relatively protected circulation. Intramucosal pH did not correlate well with DO2. Regional tissue oxygenation as measured by NIRS shows excellent correlation with global oxygen delivery. NIRS may allow estimation of systemic oxygen delivery using rapid non-invasive techniques.

Redox status of cytochrome a,a3: a noninvasive indicator of dysoxia in regional hypoxic or ischemic hypoxia.

Multiwavelength near infrared (NIR) spectrophotometry can monitor the redox state of cytochrome a,a3 (cyt a,a3) in vivo. Because cyt a,a3 is the most immediate reductant of oxygen, this technique has been proposed to evaluate tissue oxygenation. The purpose of this study was to examine the relationship between cyt a,a3 oxidation level as an indicator of dysoxia and oxygen uptake (VO2) when oxygen delivery (DO2) was progressively lowered in an in situ vascularly isolated hindlimb. Prospective, randomized, laboratory study. University research laboratory. Fourteen pigs. Measurement of critical values for both VO2 and cyt a,a3 oxidation during ischemic and hypoxic hypoxia. The right hindlimb of anesthetized, paralyzed, and ventilated pigs was subjected to progressive ischemic or hypoxic hypoxia for 100 mins by ten stepwise decreases in DO2. In ischemic hypoxia (n = 7), arterial inflow (Q) from a pump-membrane oxygenator system was lowered from 50 to 0 mL/min, with PaO2 maintained at 100 mm Hg. In hypoxic hypoxia (n = 6), PaO2 was lowered from 100 mm Hg to 0 mm Hg. Hindlimb DO2 was calculated as the product of Q and arterial oxygen content, and VO2 as the product of Q and arteriovenous difference. The cyt a,a3 oxidation level was measured every 10 secs with a four-wavelength spectrophotometer. These parameters were measured 9 mins after each change of DO2. Critical values for both VO2 and cyt a,a3 oxidation level as a function of DO2 were determined in each animal by dual linear regression analysis. In ischemic and hypoxic hypoxia, a strong correlation was found between cyt a,a3 oxidation level and VO2 in both ischemic and hypoxic hypoxia (r2 =.90 and .87, respectively). Hindlimb vascular resistance increased in ischemic hypoxia and decreased in hypoxic hypoxia when DO2 reached critical DO2. From these results, we concluded that monitoring the cyt a,a3 redox state by NIR spectrophotometry is, in this experimental setting, a sensitive indicator of dysoxia during regional hypoxic or ischemic hypoxia.

Differential sympathetic neural control of oxygenation in resting and exercising human skeletal muscle.

Metabolic products of skeletal muscle contraction activate metaboreceptor muscle afferents that reflexively increase sympathetic nerve activity (SNA) targeted to both resting and exercising skeletal muscle. To determine effects of the increased sympathetic vasoconstrictor drive on muscle oxygenation, we measured changes in tissue oxygen stores and mitochondrial cytochrome a,a3 redox state in rhythmically contracting human forearm muscles with near infrared spectroscopy while simultaneously measuring muscle SNA with microelectrodes. The major new finding is that the ability of reflex-sympathetic activation to decrease muscle oxygenation is abolished when the muscle is exercised at an intensity > 10% of maximal voluntary contraction (MVC). During high intensity handgrip, (45% MVC), contraction-induced decreases in muscle oxygenation remained stable despite progressive metaboreceptor-mediated reflex increases in SNA. During mild to moderate handgrips (20-33% MVC) that do not evoke reflex-sympathetic activation, experimentally induced increases in muscle SNA had no effect on oxygenation in exercising muscles but produced robust decreases in oxygenation in resting muscles. The latter decreases were evident even during maximal metabolic vasodilation accompanying reactive hyperemia. We conclude that in humans sympathetic neural control of skeletal muscle oxygenation is sensitive to modulation by metabolic events in the contracting muscles. These events are different from those involved in either metaboreceptor muscle afferent activation or reactive hyperemia.

Effects of muscle contraction on cytochrome a,a3 redox state.

The relationships among mitochondrial O2 availability, O2 delivery, and lactate formation in exercising skeletal muscle remain unclear. Some data suggest that muscle O2 provision is sufficient at maximal O2 consumption (VO2max) to challenge the concept of a mitochondrial O2 limitation at VO2max. The relationships among VO2, mitochondrial O2 availability, and net lactate production were studied over a wide range of exercise intensities. Using near-infrared spectroscopy, the oxidation-reduction state of cytochrome a,a3 was monitored in the canine gracilis in vivo. Twenty adult dogs were anesthetized with alpha-chloralose, intubated, and mechanically ventilated on room air. Five-minute stimulation periods at rates of 2, 3, 4, 5, 7, 8, 10, or 12 stimuli/s were performed. VO2max generally was achieved at a stimulation rate of 8 stimuli/s; mean VO2max was 0.12 +/- 0.09 (SE) ml.min-1 x g-1. The concentration of oxidized mitochondrial cytochrome a,a3 decreased at all work loads relative to resting state and demonstrated a near-linear relationship with muscle VO2 (r2 = 0.99). Muscle lactate efflux and the lactate-pyruvate ratio also were correlated positively with cytochrome a,a3 reduction, suggesting a common regulatory mechanism coupling the processes of aerobic glycolysis and oxidative phosphorylation. At VO2max, the corresponding cytochrome oxidation was not significantly different from that observed at death. Thus, in the gracilis maximal exercise leads to near-complete reduction of cytochrome a,a3 secondary to deficient O2 provision. We conclude that VO2max is limited primarily by O2 delivery to this muscle and not by other factors limiting mitochondrial ATP production or substrate oxidation.

Failure of autoregulation after closed head injury: an experimental model.

Cats were subjected to a 3.5-atm fluid percussion impact administered to the cerebral cortex. Near-infrared spectrophotometry (NIRS) was used to measure the quantity of oxyhemoglobin and total hemoglobin in the illuminated tissue as well as the cytochrome a, a3 redox state. Corroborative data were obtained by freezing brains with liquid nitrogen and measuring cortical concentrations of ATP, creatine phosphate (CP), and lactate. Immediately postimpact there was a rise in mean arterial pressure with a 38% increase of highly oxygenated blood and a shift toward oxidation in the cytochrome a, a3 redox state. By 4 hours postimpact, cytochrome a, a3 was becoming progressively reduced despite the persistence of hyperemia. This was associated with a significant (p less than 0.01) decrease in ATP and CP concentration. Additional studies in which a 0.5-sec, 100-v electrical seizure was induced before and after fluid percussion demonstrated significant differences in seizure response, indicating a failure of autoregulation.

Potassium ion homeostasis and mitochondrial redox status of turtle brain during and after ischemia.

To account for the remarkable capacity of turtle brain to survive without oxygen, microelectrode recordings of extracellular potassium activity (K+o) were made in intact brains of normoxic pentobarbital-anesthetized turtles during and after ischemia. Changes in redox status of brain cytochrome a,a3 were recorded simultaneously by reflection spectrophotometry. Cytochrome a,a3 became fully reduced, and K+o rose at a slow rate for approximately 60 min during complete ischemia. When K+o reached 8-12 mM, an inflection point was observed after which K+o rapidly increased to levels above 30 mM. Full recovery of baseline K+o and cytochrome a,a3 redox state occurred only if brain blood flow was restored within 60 min. These data demonstrate that maintenance of ion homeostasis in turtle brain depends on blood flow and continued delivery of systemic substrates and that viability is threatened if ischemia is prolonged beyond 60 min.

Near infrared spectrophotometry: potential role during increased intracranial pressure.

Two experiments were conducted to assess the feasibility of near infrared spectrophotometry (niroscopy) to directly monitor the effects of increased intracranial pressure on brain metabolism. Intracranial pressure (ICP) was increased in cats by subarachnoid infusion of a "mock" CSF solution. Cytochrome a,a3 redox state, oxyhemoglobin, deoxyhemoglobin and cerebral blood flow were non-invasively and continuously monitored by niroscopy. The results of both experiments indicated that untreated increases in ICP correlated with a reduction in cytochrome a,a3 redox state (p less than 0.01), a decrease in the quantity of oxyhemoglobin and cerebral blood flow (p less than 0.01), and an increase in deoxyhemoglobin. This study suggests that niroscopy has the potential for providing noninvasively and continuously data assessing brain metabolic activity. The excellent correlations obtained with simultaneous direct measurements of intracranial pressure make this an attractive method for eventual application to humans at risk for increased intracranial pressure. The value of niroscopy is even more evident in Exp. II where it can be seen that knowledge only of ICP would give the physician a false sense of security, whereas direct, non-invasive, continuous assessment of brain perfusion and oxygenation may well prove to be more appropriate parameters to monitor.

Cytochrome a,a3 reoxidation. Early indicator of metabolic recovery from hemorrhagic shock in rats.

To assess the metabolic recovery of mitochondria after injury, we have monitored, in vivo and noninvasively, changes in the redox state of cytochrome (cyt) a,a3 in 35 rats after tissue hypoxia induced by rapid exsanguination to a mean arterial pressure of 30-35 mmHg. This level of mean arterial pressure was maintained for a shorter period of time in group I (n = 17) and a longer period of time in group II (n = 18), then the shed blood was returned by infusion. The surviving animals were observed for 2 more h before terminating the experiments. During exsanguination, reinfusion and recovery intervals brain tissue parameters of blood oxygenation, relative blood volume, and cyt a,a3 redox state were monitored continuously by spectrophotometry through the closed skull and intact skin. Group I had a high survival rate while group II had a very low survival rate. In both groups, with the onset of hypotension, there was a prompt rapid shift, followed by a slow continued progressive shift, of cyt a,a3 toward a more reduced state. The extent of recovery of cyt a,a3 following reinfusion was different in each group. In group I there was a rapid reoxidation of cyt a,a3 to a level above the base line (16 +/- 12%, mean +/- SEM). In contrast, the extent of reoxidation of cyt a,a3 in group II was significantly lower and stayed 31 +/- 6% below the base-line level. To further evaluate the mechanisms responsible for these observations, another related experiment was performed. 12 rats were subjected to shock and resuscitation as outlined for groups I and II. After death or killing of the animal, we measured, in vitro, oxygen consumption of cerebral cortical slices. Oxygen consumption of cortical tissue slices in subgroup I was significantly higher than in subgroup II. We conclude that, under these experimental conditions, the oxidative response of cyt a,a3 correlates closely with survival or death in the two groups. If in group I animals the greater oxidation of cyt a,a3, in vivo after resuscitation, reflects greater oxygen utilization, as is suggested by the in vitro observations in subgroup I, then we may be observing a useful adaptive response to tissue injury leading to preserved organ function and enhanced survival. Therefore, noninvasively measured cyt a,a3 redox state, reflecting intracellular metabolic activity, seems to indicate both the overall cerebral cellular response to injury and the likelihood of survival.

An evaluation of perfluorochemical resuscitation after hypoxic hypotension.

The relative performance of whole blood and saline, a balanced salt-albumin solution, and Fluosol-43 were compared in an experimental animal model of combined hypoxia and hypotension. The test fluids were evaluated in terms of their respective ability to restore mean arterial pressure and provide adequate oxygen to restore and maintain a normal brain cytochrome a,a3 redox state, restore and sustain normal cerebral cortical ATP and creatinine phosphate concentrations (CP), and return cerebral cortical lactate concentrations to normal after the hypoxic-hypotensive period. With regard to ATP, CP, lactate, and cytochrome a,a3, all three test fluids performed equally well inasmuch as there were no significant differences between groups. None of the test regimens resulted in normal ATP concentration post-hypoxic hypotension. Although CP concentrations were lower than baseline after resuscitation, the difference was not statistically significant. Fluosol-43 resuscitation resulted in a significantly higher (p less than 0.05) mean arterial pressure by 120 minutes post-resuscitation. It was concluded that Fluosol-43 is an acceptable resuscitative fluid, most beneficial at extremely low hematocrits, but that selection of Fluosol-43 over balanced salt-albumin could not be supported when post-resuscitation hematocrits were in the 20 to 25% range.

Incomplete cerebral ischemia in the rat: vascular and metabolic changes as measured by infrared transillumination in vivo.

Cerebral blood volume, hemoglobin saturation and the cytochrome a, a3 redox state were monitored simultaneously by using three wavelengths of light in the near infrared portion of the spectrum for transillumination of the intact skull of rats. The changes in these parameters following incomplete cerebral ischemia were assessed in Wistar and Long-Evans rats submitted to carotid ligation. Another group of Wistar rats was submitted to vertebral + carotid occlusion. The experiments, performed under N2O/O2 anesthesia, showed that in all three groups carotid occlusion induced a decrease in blood volume, Hb saturation and a reduction of cyt. a, a3. However, the cytochrome redox state tended to normalize during ischemia as a consequence of higher O2 extraction from blood. The primary finding of this study was the marked hyperoxidation of cyt. a, a3 which occurred after reestablishing of the carotid blood supply, in spite of a secondary post-ischemic hypoperfusion of the brain. Although uncoupling of oxidative phosphorylation cannot be excluded the dissociation between blood supply and metabolism could well be due to ischemia-induced hypermetabolism of the central nervous tissue. In view of the marked oxidation of cyt. a, a3 during the reperfusion period as compared with the small extent of its reduction during the ischemic episode, the data also support the hypothesis that under steady state conditions in vivo, cytochrome oxidase is mainly reduced.