Clyde H. Barlow

Evaluation of Cardiac Ischemia by NADH Fluorescence Photography

A direct, noninvasive method of assessing the oxidation-reduction potential of an intramyocardial respiratory chain component is described. The technique is based on the differences in spectral properties between the oxidized and reduced forms of nicotinamide adenine dinucleotide (NADH). The tissue surface fluorescence from intracellular NADH may be measured and documented photographically. Noose occlusion of a coronary artery produced detectable NADH fluorescence in 15 seconds in the subtended ischemic epicardium. This fluoresence of reduced pyridine nucleotide resolved following 60 seconds of reperfusion of the ischemic myocardium. The reduction of epicardial NADH with ischemia is a rapid and reversible process. A subsequent noose reocclusion resulted in a reproducible pattern of fluorescence. The technique of NADH fluorescence photography appears superior to current methods of assessing tissue oxygen supply:demand.

Temporal relation between onset of cell anoxia and ischemic contractile failure: Myocardial ischemia and left ventricular failure in the isolated, perfused rabbit heart

Contractile dysfunction is characteristic of the acutely ischemic myocardium. This study was undertaken to assess the temporal relations between the onset of cell anoxia and ischemic contractile failure in isolated, isovolumetric contracting rabbit hearts. High speed epicardial fluorescence photography using reduced nicotinamide adenosine nucleotide (NADH) was used to identify areas of cell anoxia. The onset of ischemia was correlated with deterioration of pressure generation over the course of sequential 60 second coronary arterial occlusions. In the isovolumetric contracting rabbit heart, areas of ischemia were detected 2 seconds after coronary occlusion. Significant reduction in peak systolic pressure occurred at 6 seconds of ischemic time and pressure continued to decrease throughout the 60 second period of coronary occlusion. NADH accumulation indicates imbalance of myocardial oxygen supply and demand and the cessation of oxygen utilization by the mitochondria. The results of this study indicate that ischemia is detectable within 1 to 2 seconds after coronary occlusion and that ischemic ventricular dysfunction occurs several seconds thereafter. Myocardial oxygen reserve is negligible.

Time resolved 3-dimensional recording of redox ratio during spreading depression in Gerbil brain

Optical fluorescence and reflectance measurements have been used to map the distribution of metabolic states in three dimensions in the gerbil brain with a spatial resolution of 200 microns an a time resolution of 4-6 s. In Mongolian gerbils anesthetized with pentobarbital, the redox states of the nicotinamide adenine dinucleotide (NADH) and flavoprotein components of the electron transport chain exhibit two distinct phases during the wave of spreading depression: (1) a transient period of oxidation and (2) a prolonged period of reduction, during which the cytochromes are reduced, and the hemoglobin is predominantly in the deoxy form. These data are interpreted as indicating that the energy demand placed on the gerbil brain during such spreading depression wave is sufficient to drive the brain temporarily hypoxic.

Fluorescence of pyridine nucleotide and flavoproteins as an indicator of substrate oxidation and oxygen demand of the isolated perfused rat kidney

Abstract To evaluate the relationship between renal function and mitochondrial redox state the followng photometric techniques were applied to the isolated perfused rat kidney and correlated to changes of experimental conditions: 1. 1. Surface fluorescence of reduced pyridine nucleotides (PN) and oxidized flavoproteins (FP) was recorded simultaneously with a double beam fluorometer. 2. 2. PN-fluorescence was monitored continuously with a DC-fluorometer. 3. 3. Photographs of PN-fluorescence were taken with a large format camera. The results led to the following conclusions: 1. 1. Since a close relationship exists between FP- and PN-fluorescence during normoxic-anoxic transitions, PN-fluorescence of renal cortex reflects mainly the mitochondrial redox state, while the cytoplasmic signal seems of minor importance. 2. 2. In spite of the high flow rate and the high O 2 -saturation of the perfusate, renal function during isolated perfusion with dextran is limited by O 2 -delivery to tissue, as demonstrated by the more oxidized mitochondrial redox state at low perfusion temperatures (28, 20, 14°C) compared to 38°C. 3. 3. Calculated on the base of PN-reduction l -lactate seems to be more utilized for oxidative phosphorylation of kidney cortex than l -glutamine. d -glucose had no effect on PN-fluorescence indicating that d -glucose is not oxidized in renal cortex in large amounts. This finding is supported by the fact that during perfusion with d -glucose in the absence of other substrates renal function was not different from substrate-free perfusion. 4. 4. The PN-fluorescence photographs show the repetitive pattern characteristic for tubular organisation at the kidney surface demonstrating morphological and metabolic heterogeneity.

CORRELATION OF ELECTRICAL SIGNALS AND MITOCHONDRIAL REDOX STATE DURING SPREADING DEPRESSION

Publisher Summary This chapter discusses the correlation of electrical signals and mitochondrial redox state during spreading depression. Spectroscopic experiments indicate that the cerebral blood supply in pentobarb anesthetized gerbils is insufficient to supply the oxygen needed for the increased cellular metabolism induced by the phenomenon of spreading depression of Leao (SD). DC electrical signals recorded from a single point on the cortex of anesthetized animals, following initiation of SD, show a biphasic change that correlates with a wave in which intracellular potassium ions are released to the extracellular space. The studies of brains freeze-trapped during SD show an annulus of tissue in which fluorescence measurements demonstrate that the mitochondrial NADH and flavoproteins are completely reduced and in which reflectance spectroscopy shows the hemoglobin to be deoxygenated. The interpretation of this data is that the increased metabolism needed to repump the K+ ions is sufficiently high so that the oxygen demand exceeds supply, and the mitochondria drive themselves into a mitochondrial state. The intense, complete mitochondrial reduction during SD can be decreased by making the animals hypercapnic. The vasodilation of the cerebral blood vessels allows increased oxygen supply to the metabolically active region.

ORIENTATION OF THE NO LIGAND OF CYTOCHROME a3 IN NITROSYL CYTOCHROME c OXIDASE

Investigations of the site of oxygen reduction in cytochrome c oxidase have recently been accelerated by the development of techniques to orient ‘membranous’ cytochrome c oxidase [ 1,2 1, submitochondrial particles [3,4] and mitochondria [S,6] into hydrated multilayer arrays. The orientation of the heme prosthetic groups relative to the membrane plane has been determined for several oxidation and liganded states of cytochrome c oxidase and for other members of the mitochondrial electron transport chain [l-7]. This work extends these studies to the orientation of the dissociable NO ligand of cytochrome a3 in nitrosyl cytochrome c oxidase. Preliminary account of this work has appeared already [S] .

Decrease in acute myocardial ischemia by hyaluronidase in isolated, perfused, rabbit hearts☆

Hyaluronidase has been reported to reduce infarct size and cellular damage after coronary artery occlusion. The influence of hyaluronidase (H) on experimental myocardial ischemia was studied in isolated perfused rabbit hearts. Changes in ischemic area were assessed by epicardial NADH fluorescence photography, an intrinsic, high-resolution display of myocardial ischemia. Computerized determination of ischemic area was made from standardized photographs. H was begun 5 min after coronary artery ligation at 4 units/ml perfusate. NADH fluorophotographs were taken at 10-min intervals up to 40 min of ischemia. Coronary sinus oxygen tension (PesO2) myocardial oxygen consumption (MV O2) and coronary flow were determined. After 40 min, the hearts were perfused with a rhodamine solution to indicate areas of myocardial perfusion. In 18 H-treated hearts 55 ± 6% (mean ± SE) of the nonperfused area was ischemic (NADH fluorescent) and the ischemic areas had a patchy distribution. In 26 untreated hearts 86 ± 2% of the nonperfused area was ischemic and the ischemic areas were uniform, (P < 0.001). The distance between perfused and ischemic tissue was 861 ± 76 μm in the H-treated and 359 ± 19 μm in the untreated hearts, (P < 0.001). In the H-treated hearts PesO2 increased to 154% of the postligation control while it decresed to 77% in the untreated hearts (P < 0.001). MV O2 decreased to 87% of control after ligation in both groups. The H-treated hearts had a further decline of 37% while the untreated hearts had no further change. In the H-treated hearts, coronary flow increased to 150% of the postligation control while it fell to 80% in the untreated group (P < 0.001). We conclude that hyaluronidase increases PesO2 and coronary flow while it decreases MV O2 during acute ischemia. In hyaluronidase-treated hearts, significant amounts of myocardium remain normoxic within the nonperfused areas.

REDOX GRADIENTS IN OXYGEN DELIVERY TO TISSUE

Publisher Summary This chapter discusses redox gradients in oxygen delivery to tissue. The considerable developments of nondestructive techniques for measuring the amount of oxygen needed for tissue respiration include a technological progression from Warburgs manometer studies of the P50 of yeast cells to current spectroscopic and fluorometric recordings of surface and three-dimensional redox studies of cells, tissues, and organs. These developments are capped by the measurement of bioenergetic output of mitochondria by NMR determinations of the creatine phosphate/phosphate ratio for organs in vivo in a completely noninvasive way. The oxygen tensions that activate energy coupling are determined from the titrations of suspensions of isolated mitochondria under conditions appropriate to the measurement of very low oxygen concentrations. The dilution technique is preferred, whereby a small volume of standard oxygen solution is diluted several thousand times into a larger volume of suspended mitochondria. The bioenergetic function of the mitochondria, calcium uptake, and ATP formation and the generation of membrane potential are assayed independently under such conditions.