NADH brain surface scanning and 3-D computer display

doi:10.1016/0006-8993(74)90823-3

Brain Research

Volume 76, Issue 3, 23 August 1974, Pages 473-479

https://doi.org/10.1016/0006-8993(74)90823-3 Get rights and content

Abstract

The method of NADH oxidation-reduction state microfluorometry has been applied to the surface of the rat brain in an attempt to record a two-dimensional picture of NADH fluorescence in normoxia, with the animal breathing 20% oxygen, and to determine the extent and location of fluorescence intensity changes in hypoxia caused by lowering inspired oxygen level to 2%.

The 50 μm beam of a microfluorometer was scanned in parallel lines 250 μm apart over the cerebrocortical surface, dura intact. Chart records of corrected fluorescence, or 366 nm reflected light substracted from 450 nm fluorescence emission in a 1:1 ratio, were digitized and the resulting data utilized by a computer to produce ‘three-dimensional’ plots of normoxic and hypoxic fluorescence and the difference between them.

Heterogeneities were noted in the distribution of fluorescence over the cortex in normoxia. Non-uniformity of changes in local fluorescence magnitude with hypoxia was also observed.

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The adaptation of a commercially available fluorescence microscope for continuous monitoring of endogenous NAD(P)H levels in brain slices is described. A specially designed self-stirring perifusion chamber was used to monitor a stable fluorescence signal for hours, indicating sufficient oxygen and substrate supply under measuring conditions. Profiles and reversibility of the fluorescence signals following changes in NAD(P)H levels in the picomolar range are demonstrated in slices of rat hippocampus. The degree of reduction obtained under normal metabolic conditions in the presence of 10 mM glucose was 29.9% ± 1.3 (mean ± S.D.; n = 12). The present technique allows convenient monitoring of the influence of various exogenous factors on metabolic conditions which are accompanied by changes in the redox state of brain nicotinamide nucleotides.
Brain NADH redox state monitored in vivo by fiber optic surface fluorometry
1984, Brain Research Reviews
A new approach for the evaluation of brain energy metabolism in awake animals became possible as UV transmitting optical fibers became available. A variety of surface fiber optic fluorometers/reflectometers which were developed during the past decade enabled the monitoring of intramitochondrial NADH redox state in unanesthetized animals. The bundle of flexible fibers was connected to the brain via a cemented light guide holder implanted epidurally. The two signals obtained, 366 nm reflectance and 450 nm fluorescence, are subjected to various artifacts not connected to the intramitochondrial NADH redox state. In our system, the effects of movement artifacts and changes in blood oxygenation are negligible while the effects of tissue absorption or blood volume changes are considerable and could be minimized by subtraction of the two signals (1:1 ratio) providing the corrected fluorescence signal.
The brain was exposed to various physiologcal and pathological conditions which resulted in the increase or decrease in the level of NADH. Under anoxia, hypoxia and ischemia, oxygen availability decreased and the metabolic state of the brain became more reduced (state 4–5 transition). When the brain was activated by seizures, spreading depression of hyperbaric oxygenation NADH became more oxidized (state 4-3 transition).
Oxygen dependence of dopamine-β-hydroxylase activity and lactate metabolism in synaptosomes from rat brain
1982, Brain Research
Dopamine-β-hydroxylase activity can be assayed in vitro in suspensions of rat hypothalamic synaptosomes by following tritium release from [2-³H]dopamine in the presence of monoamine oxidase inhibitors. The activity is inhibited by diethyldithiocarbamate, fusaric acid, amphetamine, reserpine, and desipramine. It is not sensitive to addition of tyrosine at the concentration present in cerebrospinal fluid, to added fumarate or to a rat heart extract known to contain potent inhibitors of the isolated enzyme. The reaction in air follows simple saturation kinetics with respect to variation in dopamine concentration with aK_m of 0.075 μM. There is reversible inhibition of activity when it is assayed at low oxygen tension. The apparent oxygen affinity is such that the enzyme does not appear saturated with respect to oxygen even at arterial oxygen tensions. Other reactions assayed exhibit less sensitivity to hypoxia: production of lactic acid from glucose becomes stimulated at 10–12 mm Hg; and oxidation of lactic acid is not inhibited at oxygen tensions above 3–4 mm Hg. Comparison of these data with the distribution of oxygen tensions present in brain suggests a basis for classifying oxygen dependent neurochemical reactions.
Two-dimensional analysis of the redox state of the rat cerebral cortex in vivo by NADH fluorescence photography
1977, Brain Research
A photographic method for measuring two-dimensional changes in NADH fluorescence and hemoglobin distributions in the rat cerebral cortex in vivo has been developed. Intracellular NADH was excited by UV light peaking at 360 nm and the emission was observed through a window with the maximum transmission at 450 nm. The fluorescence photographs (360 → 450nm) required 20–25 sec exposures at the aperture opening of f/5.6 and the reflectance photographs (360 → 360nm) 10 sec exposures at f/32. The digitization of photographic images was achieved either by a PDP-8-controlled microdensitometer coupled to an A/D converter or by a combination of a manually operated microdensitometer and a computer-controlled digitizer. In the latter case, a photographic negative was scanned with a Joyce-Loebl microdensitometer in parallel lines 170 μm apart, and the densitometric tracings were digitized with a PDP-8-controlled TV digitizer. The digital data were processed by DEC PDP-10 computer and the results were displayed in 3-dimensional surfaces.
Nitrogen anoxia caused increases in fluorescence at 450 nm ranging from 10 to 75% of the normoxic fluorescence intensities (after correcting for the logarithmic characteristics of the photographic films) and decreases in reflectance intensities in the range of 10–30%. The spatial resolution of the present technique is limited to approximately 30 μm × 30 μm on the cortical surface and the time resolution to 10–25 sec. The optical properties of the cerebral cortex in vivo appear to be controlled primarily by blood vessel patterns and hemodynamic factors and secondarily by the redox state of the tissue. Evidence for a heterogeneous redox response of the cerebral cortex toward N₂ anoxia was obtained.
Cortical oxygen consumption and NADH fluorescence during metrazol seizures in normotensive and hypotensive cats
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The relationship between the oxidation of intramitochondrial NADH and increased oxygen utilization previously described in various preparations has not yet been directly studied in large areas of the cerebral cortex during generalized seizures. In the present study, changes in the NADH fluorescence of one or both cortical hemispheres were measured with a television fluorometer during Metrazol seizures in normotensive and hypotensive states. Fluorescein fluorescence was used as a reference signal. Combined with measurements of sagittal sinus blood flow and oxygen saturation, this technique demonstrated generalized ictal decreases in NADH fluorescence, the time integrals of which linearly related to the integrals of simultaneous increases in relative cortical O₂ consumption during seizures occurring at normal blood pressures. Seizures observed during systemic hypotension and probable cerebral hypoperfusion, however, were characterized by generalized increases in NADH fluorescence, the integrals of which were not related to the integrals of relatively delayed increases in cortical O₂ consumption. Similar anomalous NADH increases during seizures were also occasionally seen in certain cortical areas in normotensive animals, suggesting local ischemia. In addition. NADH fluorescence and cortical O₂ consumption were found to be affected by changes in blood pressure in the absence of ictal activity. These observations suggest that cortical NADH fluorescence is dependent both on metabolic demand and O₂ availability, and consequently may be validly utilized as an indicator of activated cortical oxidative metabolism during seizures only under conditions of adequate cortical oxygenation.
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NADH brain surface scanning and 3-D computer display

Abstract

Europ. J. Cancer.

Brain Research

Localization fluorometry of oxidation-reduction states of intracellular pyridine nucleotide in brain and kidney cortex of the anesthetized rat

Science

Intracellular oxidation-reduction statesin vivo

Science

Biochemical studies of transitions from rest to activity

Basic principles of tissue oxygen determination from mitochondrial signals

Oxygen tension in mammalian brain