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INVITED REVIEWS
The Mina and Everard Goodman Faculty of Life Sciences and the Leslie and Susan Gonda Multidisciplinary Brain Research Center, Bar-Ilan University, Ramat-Gan, Israel
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ABSTRACT |
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 TOP ABSTRACT SPECTROSCOPIC MONITORING OF...SCIENTIFIC BACKGROUND AND...FACTORS AFFECTING NADH...NADH UNRELATED FACTORSMONITORING HUMAN BODY ORGANSMONITORING NADH AND THE...SUMMARY AND CONCLUSIONSREFERENCES |
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real-time tissue viability; tissue spectroscopy; patient monitoring
Since the discovery of pyridine nucleotides by Harden and Young (94), >1,000 papers have been published on the use of NADH (Fig. 1A) as a marker for mitochondrial function. In 2000, Schleffler et al. (217) reviewed mitochondrial research methods over the past century. A major aspect of mitochondrial function, namely monitoring the energy state of tissues in vivo, was not discussed in that review. Therefore, the present review will summarize 50 years of research, started in 1955 by Chance and Williams (56, 57), by defining the mitochondrial metabolic state in vitro. To understand mitochondrial function in vivo and under various pathophysiological conditions, it is important to monitor the redox state of the respiratory chain in real time. The present review will discuss the monitoring principles for one of the electron carriers, namely, nicotinamide adenine dinucleotide (NADH). It is well known that mitochondrial dysfunction is involved in many diseases, such as ischemia, hypoxemia, Parkinson's disease, Alzheimer's disease, and in the apoptotic process. Therefore, the possibility of monitoring the mitochondrial NADH redox state in experimental animals and patients is of great importance.
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To assess the energy demand, it is necessary to measure different organ-specific parameters. In the brain, the energy demand can be evaluated by measuring the extracellular levels of K+ that reflect the activity of the major ATP consumer: Na+-K+-ATPase (152, 161). In the heart, most of the energy is consumed by the muscle contraction activity. On the other hand, the energy supply mechanism is the same in all tissues: oxygenated blood reaching the capillary bed releases O2 that diffuses into the cells. Therefore, it is possible to evaluate tissue energy supply by monitoring the same four different parameters in all tissues.
The main function of the mitochondria is to convert the potential energy stored in various substrates (e.g., glucose) into ATP. The inner membrane of the mitochondria contains 5 complexes of integral membrane proteins, including NADH dehydrogenase (complex 1). Three of those proteins are involved in the respiratory chain activity. The main function of the respiratory chain is to gradually transfer electrons from NADH and FADH2 (originating from the TCA cycle) to O2. With the addition of protons (H+), H2O is generated in complex 4. NADH (Fig. 1A, right side) is a substrate or a coenzyme for the enzymatic activity of dehydrogenases that form part of the respiratory chain and reside in the inner membrane of the mitochondria. Further details on the biochemical properties of NADH can be found in various publications (55).
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SPECTROSCOPIC MONITORING OF NADH: AN HISTORICAL OVERVIEW |
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TOPABSTRACTSPECTROSCOPIC MONITORING OF...SCIENTIFIC BACKGROUND AND...FACTORS AFFECTING NADH...NADH UNRELATED FACTORSMONITORING HUMAN BODY ORGANSMONITORING NADH AND THE...SUMMARY AND CONCLUSIONSREFERENCES |
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Because the oxidized form NAD+ does not absorb light in this range, it was possible to evaluate the redox state of the mitochondria by monitoring the UV absorbance (see Monitoring UV absorbance by NADH) or blue fluorescence of NADH (see Monitoring NADH fluorescence).
Undoubtedly, the pioneering work of Britton Chance of the Johnson Research Foundation at the University of Pennsylvania in Philadelphia led to the establishment and development of the unique measurement technology and theoretical conceptualization of the mitochondrial function based on NADH redox state monitoring in vitro as well as in vivo.
The foundations for future NADH monitoring in vitro and in vivo were established mainly in the 1950s; thus this period will be discussed in this section.
Monitoring of NADH UV absorbance
In 1951, Theorell and Bonnichsen found a shift in the absorption spectrum of DPNH upon addition of alcohol dehydrogenase (238). In the same year, Theorell and Chance described a new spectrophotometric technique for measuring the formation and disappearance of the compound of alcohol dehydrogenase and NADH (239). In 1952, Chance showed the applicability of this new technique to the measurements of pyridine nucleotide enzymes of muscle homogenate or intact cells (25). In 1954, Chance and Williams briefly described new sensitive differential spectrophotometric methods applied to the study of reduced NADH in isolated rat liver mitochondria and the same approach was used by Connelly and Chance (61) in monitoring NADH in stimulated frog nerve and muscle preparations. The oxidation of NADH in the muscle was similar to its oxidation in isolated mitochondria upon addition of ADP. In a comprehensive paper, "Enzyme mechanisms in living cells," Chance described in detail the measurements of the respiratory enzymes, including NADH (26).
A major milestone in NADH monitoring was the technique presented in 1954 by Chance (27) using a double beam spectrophotometer to determine the appropriate wavelengths in measurements of respiratory enzymes.
The detailed descriptions of the respiratory chain and oxidative phosphorylation in the mitochondria (published in 1955 by Chance and Williams) established our basic knowledge of the mitochondrial function (57). Chance and Williams defined, for the first time, the metabolic states of isolated mitochondria in vitro, depending on the substrate, oxygen, and ADP levels. In addition, they correlated those metabolic states to the oxidation-reduction levels of the respiratory enzymes. The physiological significance of those metabolic states was discussed in 1956 by Chance and Williams (58).
Monitoring NADH fluorescence
The fact that NADH was monitored by the difference in the absorption spectrum of its reduced form, limited the use of that technique to the study of mitochondria in vitro, and in very thin tissue samples (e.g., muscle) or in cell suspension. To provide a method more specific than absorption spectroscopy, fluorescence spectrophotometry in the near-ultraviolet range was applied for NADH measurement. The initial model of fluorescence recorder was described by Theorell and Nygaard in 1954 (240). The first detailed study using fluorescence spectrophotometry of NADH in intact Baker's yeast cells and algae cells was published in 1957 by Duysens and Amesz (75).
In the next 5 years (1958–1962), the monitoring of NADH fluorescence was significantly expanded, led by Chance and collaborators. In a first preliminary study, Chance et al. (37) performed simultaneous fluorometric and spectrophotometric measurements of the reaction kinetics of bound pyridine nucleotides (PN) in the mitochondria. In the same year (1958), Chance and Baltscheffsky presented preliminary results of measuring the fluorescence of intramitochondrial PN (34). In this study, they proved the connection between the mitochondrial metabolic state and the redox state of NADH as measured by spectral fluorometry in mitochondria isolated from rat liver (57). The correlation between the enzymatic assay of PN and sensitive spectrophotometry was investigated by Klingeberger et al. (120) by using the rat liver, heart, kidney, and brain.
In 1959, Chance and collaborators were able to expand the use of NADH fluorometry to various experimental models, from isolated mitochondria to intact tissue. To monitor NADH localization in intact cells, Chance and Legallais (42) developed a unique differential microfluorimeter with a very high spatial resolution. This approach was used in various cells to identify the intracellular localization of NADH fluorescence signals (54, 201). The next step was to apply the fluorometric technique to the higher organization level of animal tissues. Together with Jobsis, Chance measured in vitro changes in muscle NADH fluorescence following stimulation (41). In another paper published by Chance and Theorell (55) the authors came to the very significant conclusion that "The oxidation and reduction state of mitochondrial pyridine nucleotide without a measurable change of cytoplasmic fluorescence suggest that compartmentalization of mitochondrial and cytoplasmic pyridine nucleotide occurs in vivo, at least in the grasshopper spermatid."
An intensive use of the in vivo NADH monitoring approach started in 1962. The "classic" paper on in vivo monitoring of NADH was published in 1962 by Chance et al. (36). They were able to simultaneously monitor the brain and kidney of anesthetized rats using two microfluorometers. In 1962, Chance and collaborators elaborated on this kind of in vivo monitoring and used it in other rat organs (43, 50).
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SCIENTIFIC BACKGROUND AND TECHNOLOGICAL ASPECTS |
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TOPABSTRACTSPECTROSCOPIC MONITORING OF...SCIENTIFIC BACKGROUND AND...FACTORS AFFECTING NADH...NADH UNRELATED FACTORSMONITORING HUMAN BODY ORGANSMONITORING NADH AND THE...SUMMARY AND CONCLUSIONSREFERENCES |
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The biochemical and physiological significance of these spectral qualities is also universally accepted, that is, an increase in the fluorescence intensity indicates a more reduced state of NADH and of the rest of the mitochondrial electron transfer chain. Under various circumstances, changes in the redox state of the electron transport chain can be associated with various conditions.
To monitor NADH fluorescence, it is possible to use one of the two principles available. At the early stage, it was necessary to measure and identify the fluorescence spectrum of NADH. Fluorescence spectra were compared in different in vitro and in vivo preparations. In parallel, the second approach was adopted, namely, measuring the total fluorescence signal accumulated and integrated into a single intensity using appropriate filters. This approach was necessary to measure NADH fluorescence continuously. The following parts of this section describe the fluorescence spectra of NADH measured in various in vitro and in vivo models by different investigators. We present this review of the reported spectra to describe the foundations for the second monitoring approach, namely, the continuous monitoring of integrated spectra.
Fluorescence Emission Spectra of NADH
NADH in solution.
Several investigators have measured NADH fluorescence in solution. Very recently, Alfano's group (62) performed a calibration test of pure 
-NADH in solution, compared it to porcine myocutaneous flap, and found a very significant correlation. The NADH solution spectrum and mitochondrial spectrum were also compared by Chance and Baltscheffsky (34).
Similar spectra of NADH in solution were recorded by Schomacker et al. (219) using 337-nm excitation light for colonic tissue diagnosis.
NADH spectra in isolated mitochondria. The excitation and emission spectra of NADH (PN) and flavoprotein were measured in frozen samples of pigeon heart mitochondria (52). Using rat liver mitochondria, Chance and Baltscheffsky (34) measured the fluorescence spectra in the three metabolic states defined by Chance and Williams (58). The 330-nm light excitation resulted in a fluorescence peak at 440–450 nm. The same kind of spectra was obtained by other investigators using different fluorometers or mitochondria isolated from various organs. Galeotti et al. (87) measured similar spectra from rat liver mitochondria. Using Rhodamine B as an internal standard for system calibration, Koretsky and Balaban (125) found the same spectra emitted from isolated rat liver mitochondria. Koretsky et al. (126) compared the emitted spectrum from heart homogenates (similar to isolated mitochondria) with that of dissolved heart homogenates (126).
Intact cells. The use of microfluorimetry to study intact cell metabolism was described in several publications by Kohen and collaborators (see, for example, Ref. 123).
The typical NADH fluorescence spectrum was measured in suspension of ascite tumor cells (87). This study demonstrated that the spectrum of intact cells was similar to that of NADH solution.
Using isolated myocytes, Eng et al. (78) compared the spectra measured under various conditions of the mitochondria. They found that cyanide induced an increase in the spectrum difference, whereas FCCP, used as a typical uncoupler of oxidative phosphorylation, produced a marked decrease in the spectrum.
Tissue slices and blood-free perfused organs. The next step in the development of NADH fluorometry, after its use in isolated mitochondria and single cells, was to apply it to tissue slices and isolated perfused organs in vitro.
The classic results of Chance et al. (36) were obtained from NADH in solution, in a suspension of mitochondria and in a perfused kidney slice. Only a very small spectral difference was found between the kidney slice, isolated kidney mitochondria, and the solution of NADH. The same type of comparison was performed by Aubert et al. (7) using a slice of the electric organ isolated from an electrophorus. The spectrum from the electric organ was very similar to pigeon heart mitochondria and NADH in solution. Chance (31) published more spectra originating from perfused rat heart exposed to various metabolic perturbations. A large, significant increase in the emission spectrum was recorded between normoxic and anoxic conditions, and between normoxic and amobarbital (Amytal)-treated hearts. These results suggested that when O2 supply is eliminated (state 5) or when site I in the mitochondria is blocked by Amytal, a large change in NADH levels is recorded.
The same kind of normoxic-anoxic transition was found by Jamieson and Van den Brenk (103) using isolated gastrointestinal mucosa. The studies of Chance and collaborators were further expanded to isolated rat skin. The spectrum of the skin slice was very similar to that of the liver slice and NADH solutions. To test the responsiveness of the skin to metabolic manipulations, they compared spectra under normoxic and anoxic conditions. They also found a clear, significant increase in the skin spectrum under Amytal treatment (response not shown). Another organ of interest tested by Chance and Lieberman (46) was rabbit cornea (frozen) compared with rat liver mitochondria (states 2 and 4).
Other research groups adopted NADH fluorometry and tested the validity of its principles. For example, Koretsky et al. (126) employed rapid-scan video fluorometry, and found the same NADH spectrum when using excitation light from a N2 laser (337 nm). Very recently, the spectrum of the hippocampal slice in vitro was measured by Perez-Pinzon et al. (200) and a marked increase in NADH spectrum was recorded under anoxia.
Organs in vivo. The use of NADH fluorometry for in vivo studies started at the end of the 1950s and has been presented in various publications. In a summative study, Chance compared spectra of NADH measured from rat kidney cortex to those of rat brain cortex and found that the main effect of anoxic transition, both in the kidney and the brain, was a large increase in the fluorescence intensity with no detectable shift in the spectra (36). Similar results were obtained by Chance (31) when intact sartorius toad muscle was stimulated. Stimulation of the muscle (state 4 to state 3 transition) led only to a decrease in the intensity of the fluorescence emission spectrum.
In 1975, Sundt and Anderson (232) applied in vivo fluorometry to study the brain of the squirrel monkey and recorded a greatly increased intensity in the dead brain as opposed to normal brain, with intermediate values in the ischemic brain. At the same time, Harbig et al. showed a clear increase in the NADH spectrum between normoxic and anoxic conditions in cat brain (93).
Corderio et al. (62) compared the excitation spectra in porcine myocutaneous flap with those of NADH in solution. Exposing the flap to 6 h of ischemia dramatically increased the intensity of 450 nm light. Furthermore, we compared two sources of excitation light: the usual mercury arc and laser excitation at 324 nm (152). The main problem in applying such spectra, under in vivo conditions, is the effects of hemodynamic changes under anoxia. To overcome this problem, we induced repetitive cycles of anoxia that suppress the autoregulatory compensation mechanisms (152).
Figure 1C shows six scans obtained from the normoxic (A1, A2) and the anoxic brain (B1, B2) using a Hg arc, and comparative scans using Hg (C1) and the laser source (C2) in the dead brain. The results indicate that the location of the emission spectrum peak is identical in normoxia and anoxia, and is close when the laser is used. We also used the laser source in several in vivo studies and found that the light intensity did not harm the brain during several hours of measurement. Under anoxia, a clear increase in NADH fluorescence spectra was recorded.
Comparison between NADH fluorescence intensity and biochemical analysis of pyridine nucleotides
To verify the source of NADH fluorescence signals monitored in vivo, it was necessary to freeze the monitored tissue and to biochemically measure its pyridine nucleotide content. Chance et al. (51) compared the fluorescent and analytical measurements of NADH and NADPH using in vivo liver. It became clear that ischemia, limiting the availability of oxygen, led to a large increase in the fluorescent signal. At the same time, NADH showed a marked increase while NADPH remained unchanged. These results suggested that the source of the in vivo fluorescent signal is mainly from NADH. In 1966, Chance (31) summarized the studies comparing the fluorescence signals with the biochemical analysis of pyridine nucleotides. NADH was found to be the main source of fluorescence change in the in vitro beating heart. This held true under anoxia and under treatment with Amytal. Chance et al. showed a clear correlation between the fluorescence signal measured in the heart and the biochemical analysis of NADH in the tissue.
To test conditions where NADH becomes oxidized, the hyperbaric oxygenation effect was tested in the rat liver in vivo (40). NADH was found to be the main source for the increase of in vivo signal under Amytal treatment. Under hyperbaric hyperoxic conditions, the contribution of NADPH was larger. In the same study, Chance et al. (40) showed that in the brain cortex under anoxic or hyperbaric conditions, NADH was the main source of the fluorescence signal. In 1971, Jobsis et al. (110) compared the corrected NADH fluorescence signal (after subtracting the reflectance signal) to its concentration measured in a brain tissue sample after induction of convulsions or anoxia. A very clear correlation was discovered, but Jobsis et al. concluded, "a quantitative interpretation of the changes of fluorescence in terms of nanomoles of NADH and NADPH is not truly warranted at this point." Other investigators performed similar comparisons between the fluorescence signals and the analytical measurements. Shimazaki et al. (222) showed a good correlation between the fluorescence of NADH and its concentration in control cornea as well as in cyanide treated cornea. NADH fluorescence and enzymatically determined NADH levels were similarly compared in the brain subjected to ischemia (256). Toth et al. (248) found a clear correlation between the two parameters under various metabolic perturbations of the spinotrapezius muscle. In the in vivo muscle, cyanide treatment and ischemia caused an increase in both parameters while the glycolysis inhibitor IAA induced a decrease in both. The same effects have been found recently by Toth et al. (247) in tests of cat sartorius muscle in vivo. A significant correlation was found between the fluorescence signal change in ischemia and the amount of NADH determined enzymatically.
Intracellular origin of the NADH fluorescence signal
The intracellular localization of the signals measured from NADH had been discussed even before NADH fluorescence was measured. It is important to understand that the excitation and emission wavelengths of NADH (350 and 460 nm) are well separated from other endogenous chromophores, as described by Anderson-Engels and Wilson (4) and the 460-nm fluorescent emission originates primarily from NADH bound to mitochondria.
Klingenberg et al. (120), who used mitochondria from various organs, found that the sum of NAD and NADH was of the same order of magnitude in the liver, heart, kidney, and brain. Using mitochondria of rat liver and kidney, Avi-Dor et al. (10) came to the following conclusions: "The average fluorescence yield of reduced pyridine nucleotide in mitochondria is 6 to 8 times the yield of NADH and NADPH in aqueous solution." "The yield of mitochondrial NADH is substantially higher than the yield of mitochondrial NADPH."
The significance of these conclusions is that the in vivo fluorometric technique provides information about the redox state of NADH in the mitochondria with a negligible contribution of the cytoplasm. Estabrook arrived at the same conclusion using rat liver mitochondria, by comparing the fluorometric technique with the spectrophotometric approach (80). The same conclusion was drawn by Chance et al. (36) for rat brain and kidney in vivo, and by Jöbsis et al. (110) for cat brain induced to epileptic activity. Jöbsis and Duffield (108), using the fluorometric technique in intact toad sartorius muscle, concluded "that cytoplasmic NADH does not interfere materially with these measurements." A similar conclusion was reached by Chapman (60), positing that "mitochondrial NADH is the sole significant origin of labile fluorescence under the condition used." The same conclusion was reported later by Jobsis and Stainsby (112) for the mammalian skeletal muscle. O'Connor (193), who used in vivo monitoring of cat brain in combination with biochemical assays, stated the following: "The cortical fluorescence recorded with in vivo fluorometric techniques originated from mitochondrial NADH." The same results and conclusions were obtained by Nuutinen (190) using isolated perfused rat heart. Accordingly, "The NADH+NADPH fluorescence of the intact tissue originates largely from the mitochondria." In our own studies summarized in a review paper (152), we also confirmed the notion that most of the labile NADH signal in the brain originated from the mitochondria. Additional indirect evidence comes from other decapitation model studies performed by us (160, 259). We found that in the awake rat, the NADH fluorescence increase starts within 1 s and reaches its maximal level within <1 min. The same timing pattern has been described previously (53) when effects of decapitation were studied in anesthetized mouse brain. It has been reported (144) that after decapitation, a large enhancement of glycolysis occurs, but this may have started only several minutes after the decapitation. Lowry's findings also suggest that if NADH increases dramatically several minutes after the decapitation, it may derive from the cytosolic source. In no decapitated animal (>200 animals) did we ever find such a secondary increase in NADH fluorescence after the initial maximal increase. Eng et al. (78), studying single rat cardiac myocytes, asserted that "These data are consistent with the notion that the blue autofluorescence of rat cardiac myocytes originated from mitochondrial NADH." Very recently, Coremans et al. (63) confirmed that "The results show that the NADH fluorescence/UV reflectance ratio can be used to monitor the mitochondrial redox state of the surface of intact blood perfused myocardium."
Monitoring devices and technological aspects
Principles of NADH monitoring. As described in the introductory section, NADH can be measured by utilizing its absorption spectrum in the UV range, as well as by the blue fluorescence spectrum under UV illumination. In the early stages, NADH monitoring was based on the difference in the absorption of NADH and NAD+. At the range of 320 to 380 nm, only the reduced form; NADH absorbs light, while NAD+ does not (Fig. 1B). Therefore, when a mixture of NADH and NAD+ is illuminated in a cuvette by 320–380 nm, only NADH will affect the absorption spectrum peak at 340 nm. This property of NADH was used in the early 1950s by several investigators, as reviewed in Spectroscopic Monitoring of NADH–Historical Overview. Chance and collaborators utilized this technique to measure NADH in muscle homogenates or intact cells (25) and published many papers concerning the unique absorption spectrum of NADH.
The absorption approach is not practical for measuring NADH in a thick tissue; hence, another property of NADH was used. Since the early 1950s, fluorescence spectrophotometry of NADH has been employed in various in vitro and in vivo models. The emission of NADH fluorescence, under illumination at 320–380 nm, has a very wide spectrum (420–480) with a peak at 450–460 nm (Fig. 1C). NADH fluorescence has been identified by Chance and his collaborators as a good indicator of the intramitochondrial oxidation-reduction state (48).
Types of NADH fluorometers. Since the early 1950s, different groups have developed a number of fluorometers adapted to their specific experimental protocols. As of today, most of the groups using NADH fluorometers construct the devices in house, since no suitable commercial products are, in fact, available. During the past four decades, the workshop of the Johnson Research Foundation at the University of Pennsylvania Medical School (Philadelphia, PA), headed by Prof. Britton Chance, has manufactured a few types of laboratory instruments purchased and used by investigators (45).
The basic features of NADH fluorometers consist of the following: 1) a light source (including appropriate filters); 2) an optical path to the preparation and back to the detection unit; 3) detection and signal processing units; and 4) signal recording and storage units.
In our earlier review published in 1984, we extensively specified the light-guide-based fluorometry used in our studies (152).
The review article on in vivo NADH fluorescence monitoring, published in 1992 by Ince et al. (102) included many other technical aspects of the methodology. Nevertheless, here we will elaborate on the historical development of the various models of NADH fluorometers. We recently (155) reported on a new type of NADH fluorometer based on a very small and stable UV light source: a 375-nm light-emitting diode.
In the present review, we will not recommend the use of any type of fluorometer-reflectometer, but will rather provide the reader with extensive reference information, gathered during the years, on the construction of fluorometers. Priority will be given to articles mainly dealing with NADH monitoring in vivo, though in vitro fluorometer types will be also considered. Duysens and Amez (75) schematized the first fluorescence spectrophotometer used for intact cells. They utilized the classic light source (the mercury arc) providing a very strong band at 366 nm, even though not at the maximal NADH absorption peak (340 nm). Using a monochromator, they were able to obtain the NADH fluorescence spectrum in baker's yeast cells and photosynthesing cells. Duysens and Amaz concluded that "the fluorescence excited by 366 nm can be used for measuring reduced pyridine nucleotide in vivo".
In 1959, Chance and Legallais (42) described a differential fluorometer that heralded a new era in monitoring NADH fluorescence in vivo as an indicator of mitochondrial function. They used a microscope, serving as the fluorometer basis, with two light sources: tungsten and mercury lamps with appropriate filters. In 1959, Chance and Jobsis (41) proved that mechanical muscle activity is associated with NADH oxidation measured in excised muscle. This study was the bridge from the subcellular (mitochondria) and cellular (intact cell) monitoring approaches toward actual in vivo applications.
The first in vivo NADH monitoring device was presented in the early 1960s. At that stage, the effects of scattered light and tissue absorption due to blood were not taken into consideration when monitoring NADH fluorescence. The first detailed results of in vivo NADH fluorescence measurements were published in 1962 (36).
These classic papers described two microfluorometers that were modifications of previous designs (42, 54). This microfluorimeter type employed Leitz "Ultrapack" illumination, which had been used for many years by various groups until the appearance of UV transmitting optical fibers. To avoid movement artifacts, rats were anesthetized deeply and their heads were fixated in a special holder attached to the operation table. Numerous studies utilized the principles of the "Ultrapack" illumination system. The same instrumentation was used in other in vivo studies, including those of Chance's group (38, 43, 44, 59), Dora and Kovach's group (71, 92), Rievich's group (93), Jobsis and collaborators (108, 110, 111, 213), Gosalvez et al. (89), and Anderson and Sundt (5, 232). This is only a partial list.
Monitoring NADH fluorescence and reflectance. The effect of blood on NADH fluorescence was discussed early by Chance et al. (36). To monitor NADH in vivo, Chance's group had to avoid areas containing large blood vessels, which interfere with the emission and excitation light. The monitoring of a second channel in tissue fluorometry in vivo was reported by Chance and Legallais in 1963 (44). They showed that "changes due to the deoxygenation of oxyhemaglobin do not interfere with measurement of the time course of fluorescence changes in the tissue studies."
The addition of a second monitoring signal, namely, tissue reflectance at the excitation wavelength was reported in 1968 by Jobsis and Stansby (112). It was based on a previous model described by Jobsis et al. in 1966 (107). In two more papers by Jobsis and collaborators (110, 111), the measurement of 366-nm reflectance was used for the correction of the NADH fluorescence signal from the brain. The reflectance signal was subtracted from the fluorescence signal. The same type of instrumentation was used by various groups for the measurement of NADH in single cells (124) or in vitro preparations (13, 19).
Fiber optic fluorometer/reflectometer. To enable the monitoring of NADH fluorescence in unanesthetized animals or other in vivo preparations, a flexible means was needed to connect the fluorometer with the tested organ, for example the brain. This was achieved in 1972, when UV transmitting quartz fibers became available (Schott Jena Glass). We have used the light guide-based fluorometer for in vivo monitoring of the brain (48, 157) subjected to anoxia or cortical spreading depression. The historical development of light guide-based fluorometery-reflectometry is shown in Fig. 2. The original device functioned on the time-sharing principle (Fig. 2A), where four filters were placed in front of a two-arms light guide. Filters 1 and 3 enabled the measurement of NADH fluorescence, while filters 2 and 4 were used to measure tissue reflectance at the excitation wavelength. The reflectance trace was used to correct the NADH signal for hemodynamic artifacts, and to indicate changes in the blood volume of the sampled tissue.
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Our group developed and used the model shown in Fig. 2D in the late 1970s. This model is still being used in our laboratory to monitor the brain (148), heart (2, 114), liver (15), and kidney (178, 260), and in multisite or multiorgan monitoring (133, 156, 161).
Other groups used optical fibers to connect the monitored tissue to the fluorometer, differently than the three models shown in Fig. 2. Renault and collaborators used a light guide fluorometer for monitoring heart in vivo (206, 207). Rex and collaborators used another type of light guide fluorometer for the brain and other systems (202, 208). Microlight guides were used for in vitro and in vivo studies (104, 105, 244).
Principles of NADH artifact correction. In Factors affecting NADH fluorescence and reflectance signals, we will discuss the effects of various factors on the measurement of NADH fluorescence and tissue reflectance. To compensate for NADH unrelated factors, various approaches have been developed. In the paper published by Ince et al. (102), the various correction techniques are listed and discussed in detail. It appears that the correction technique is also dependent on the instrument configuration (13, 93). Most of the published materials are based on the 1:1 ratio, when subtracting the 366-nm reflectance from the fluorescence signal. As of today, a new approach is still lacking to compensate for non-NADH factors affecting the NADH fluorescence signal. We have found that subtracting the reflectance from the fluorescence or dividing the two parameters provides similar net NADH changes. Very recently, Bradley and Thorniley (20) published a review article dealing with the various correction techniques for tissue fluorescence. They summarized their review by the following conclusion: "even though research has been conducted into correction techniques for over thirty years, the development of a successful and practical correction technique remains a considerable challenge."
Calibration of the monitored signals. To reduce the variation between different animals in a specific protocol, a standard procedure for signal calibration was used by various investigators. Since NADH could not be calibrated in absolute NADH concentrations, it was necessary to use a calibration procedure before each experiment. Different groups had developed standard procedures for calibration.
Because the technology is rapidly changing, it is unnecessary to use the old approaches for signal calibration. Ince et al. (102) listed the various calibration procedures. As an example, we will present the calibration procedure used in our laboratory before a new computerized system was integrated in our fluorometer. The reflectance and fluorescence signals obtained from the photomultipliers (RCA 931B) were calibrated to a standard signal (0.5 V), as recently described in detail (161) by variation of photomultiplier dynode voltage obtained from a high-voltage power supply. The standard signal (0.5 V) used to calibrate the recorder was set to give a half-scale deflection on the recorder (2.5 cm) with the pen resting at midscale. The gain was increased, as required, by a factor of 2 or 4 to give 50% or 25% of the full scale correspondingly. The changes in the fluorescence and reflectance signals were calculated relatively to the calibrated signals under normoxic conditions. This type of calibration is not absolute but provides reliable and reproducible results from various animals and also among various laboratories using this approach.
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FACTORS AFFECTING NADH FLUORESCENCE AND REFLECTANCE SIGNALS |
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TOPABSTRACTSPECTROSCOPIC MONITORING OF...SCIENTIFIC BACKGROUND AND...FACTORS AFFECTING NADH...NADH UNRELATED FACTORSMONITORING HUMAN BODY ORGANSMONITORING NADH AND THE...SUMMARY AND CONCLUSIONSREFERENCES |
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The following factors may affect the two measured signals, 366-nm reflectance and 450-nm fluorescence: 1) tissue movement due to mechanical or intracranial pressure changes; 2) extracellular space events, such as volume changes or ion shifts between intra- and extracellular space; 3) vascular and intravascular events, for example, oxy-deoxy Hb changes, and blood volume changes due to autoregulatory vasoconstriction under pathological conditions; and 4) intracellular space factors, such as O2 level, ATP turnover rate, substrate availability, and mitochondrial redox state. This subject will be discussed later on.
The effects of factors 1–3 will be discussed below and factor 4 in Changes in mitochondrial NADH and metabolic state.
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NADH UNRELATED FACTORS |
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TOPABSTRACTSPECTROSCOPIC MONITORING OF...SCIENTIFIC BACKGROUND AND...FACTORS AFFECTING NADH...NADH UNRELATED FACTORSMONITORING HUMAN BODY ORGANSMONITORING NADH AND THE...SUMMARY AND CONCLUSIONSREFERENCES |
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To avoid movement artifacts in monitoring patients, we used diverse approaches. In the neurosurgical intensive care unit, we used a metal holder screwed to the skull of comatose patients (162). In the operating room, we used a floating light guide probe fixed to the head holder in neurosurgical procedures (164) or a ring used to hold retractors during abdominal operations or kidney transplantations (178).
Intra- and extracellular space events. The second factor, which can be a potential source of error in NADH fluorescence measurement, is a change in the absorption properties of the tissue, during various perturbations, at the observed site. This artifact has been recognized mainly in brain studies. Very little is known and published about this factor, due to the inability to separate it from other factors affecting the NADH fluorescence readings. It seems to us that under physiological or pathological conditions involving ions and water movement between the intracellular and the extracellular space, this factor may have a greater effect on NADH fluorescence measurements. We have earlier published indirect evidence for the involvement of this factor in our measurements and a possible correction method.
First, when the blood was eliminated from the brain, using a fluorochemically perfused brain preparation (174), only very small, if any, changes in reflectance were measured during the anoxic cycle. As shown in 
Fig. 4B, the uncorrected NADH fluorescence and the corrected fluorescence (CF) had similar kinetics. Furthermore, the CF response of the perfused brain to anoxia was similar to that of the blood-perfused brain in the same animal before the initiation of perfusion shown in Fig. 4A. However, when a spontaneous spreading depression (SD)-like response was recorded in several perfused brains, the reflectance tracing showed a change typical of the initial increase phase of the regular response to SD found in a normoxic blood-perfused brain (for details, see Ref. 174). The second phase of the decrease in reflectance during SD did not occur in the perfused brain due to the lack of blood in the system. We believe that this initial increase in reflectance was due to water and ion movement during the SD event in the perfused brain. The same type of reflectance response (an increase) was obtained when a brain slice in vitro was stimulated (142).
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Finally, in experiments where complete cerebral ischemia was induced by decapitation in rats and gerbils (259), bilateral carotid occlusion in gerbils (166), or 4-vessel occlusion in rats (163), a large reflectance increase was recorded after NADH reached its maximum level (PO2 = 0). This increase was termed secondary reflectance increase (SRI). In recent experiments, we have monitored the DC potential as well as K+ from the extracellular space, together with NADH fluorescence and 366-nm reflectance. In all cases, we found a very significant correlation between the complete depolarization occurring during complete ischemia (as identified by the DC and K+ changes) and the SRI phenomenon. In few decapitated animals we found that during the appearance of the SRI, the corrected NADH signal decreased meaning that oxidation of NADH without the availability of oxygen. It seems that the SRI is partly due to the water and ion movements during the complete depolarization, and it may also be due to a decrease in tissue blood volume as a result of a spasm of blood vessels. In several cases, the SRI was very large (significantly) and the regular correction technique was insufficient. We have found that under partial ischemia in gerbils, with SRI occurring, a decrease in blood flow and PO2 was recorded, indicating a possible massive vasoconstriction response that might be responsible for the SRI. One may argue that the continued fall in the NADH (oxidation) after the initial increase in NADH, due to the lack of O2, is due to the loss of mitochondria via apoptotic cell death or other cell death mechanisms as suggested by Riess et al. (209) or Varadarajan et al. (252) in studying the preconditioning of the heart. We are sure that this is not the case in our system. We showed that even when complete ischemia was induced in gerbils for 30 min, the NADH signals were recovered to the baseline levels and the response of the recovered brain to cortical spreading depression was identical to that recorded before the ischemia (166). These results suggest that even during 30 min of ischemia, including the SRI, the integrity of the mitochondria was not damaged.
Vascular events. These events include changes in blood oxygenation, namely in the saturation level of HbO2, as well as changes in the blood volume in the monitored microcirculation.
Blood oxygenation. Since hemoglobin (Hb) is a strong light absorber at various wavelengths, the measurements of NADH are affected by the amount of Hb in the monitored tissue. Kramer and Pearlstein (132) attempted to use the hemoglobin isosbestic point (448 nm) to correct the NADH measurement for changes in the [Hb]/[HbO] ratio. However, the attempt was unsuccessful and without any continuation, since their paper only presented preliminary results. To test the effect of Hb concentration in the tissue on NADH fluorescence spectrum, Rahmer and Kessler (204) used perfused rat liver and found that higher concentrations of Hb corresponded to a lower intensity of the fluorescence spectrum emitted from the liver.
Very recently, Coremans et al. (63), in their measurements of NADH fluorescence and light reflectance (365 nm), tested the effects of NADH and hemoglobin on the two signals in a tissue phantom model. They concluded that the ratio between the two signals (F/R) provides a good corrected signal.
In transition from oxygenated blood (HbO2) to deoxygenated blood, the absorption spectrum is different and may affect the NADH signal. The oxy-deoxy Hb transition and its effect on the measured signals have been discussed previously (48), and this effect has been shown to be negligible. Indirect studies produced the following similar results. First, in decapitation, only a small change, if at all, was measured in the reflected light, although the hemoglobin present in the measured field was rapidly losing O2. This indicates that the oxy-deoxy Hb transition has only little effect on NADH fluorescence and on the reflected light. Second, in comparing the normoxic-anoxic transition to the anoxic-normoxic one (induced by breathing N2), the different kinetics in the reflectance traces suggest that blood oxygenation does not have a significant effect on the CF, when using 366-nm reflectance changes for correction. The decrease in reflectance after initiation of anoxia has very fast kinetics (it takes <1 min to reach the minimum reflectance level). However, during reoxygenation, the CF has very fast kinetics, and it takes 5 min or more for reflectance to reach the baseline. This indicates that the oxy-deoxy transition has only little effect on the two measured signals (reflectance, fluorescence).
Finally, in our previous publications (157, 159), we have shown that during anoxia in a normal rat, a two-step decrease in reflectance occurs. First, there is a small decrease in reflectance, which is followed by a very large decrease when NADH reaches its maximum level. This secondary decrease in reflectance during anoxia did not appear if the brain had been made partially ischemic by bilateral carotid artery occlusion for 24 h. The corrected fluorescence (CF) responses, however, were about the same. Furthermore, when N2 cycles of 1 min were applied to the same rat every 10 min, we found that the reflectance responses decreased in time, while the corrected fluorescence showed the same response to anoxia (157). This occurred even though the oxy-deoxy hemoglobin change probably took place in all N2 cycles.
Blood volume changes. Blood volume changes may occur as a response to various physiological and pathological conditions. This is the main artifact in monitoring tissue NADH fluorescence, and it has been discussed by many investigators. During the past 30 years, we have used fiber optic surface fluorometry to monitor the brain exposed to various conditions, as well as other organs such as the heart (114, 189). Hence, evidence for the involvement of blood volume artifacts and their correction have been drawn from various published experiments.
First, the typical decrease in reflectance during brain anoxia can be corrected to a level of 90–100% depending on the microcirculatory pattern of the site under observation. When hemoglobin was eliminated from the brain (perfused with a fluorochemical), no blood volume changes could occur, and indeed no changes in reflectance were observed (174). In the heart, anoxia did not have a great effect on reflectance and the observed change was in the same direction as in the brain (114). In a partially ischemic brain (147) or when N2 cycles were repeated many times (157), the typical decrease inreflectanceduring anoxia was also minimal, due to the low brain capacity to increase its blood volume to compensate for low PO2.
Second, in another set of experiments, we monitored NADH during hyperbaric oxygenation of the brain (167, 177). We found that when the animal was exposed to compressed 100% O2, a large increase inreflectancewas recorded. This was explained by a decrease in blood volume in the brain due to the vasoconstrictive response of the blood vessels to high PO2. When the compression mixture contained 1.5% CO2 at 5–6 ATA, the reflectance trace showed a sharp decrease due to the vasodilation response occurring under high PCO2. The CF showed the same level of oxidation. This shows that CF can be corrected for blood volume changes induced by high PO2 or high PCO2 in the brain, using the 1:1 subtraction technique.
Third, in studying the effects of unilateral or bilateral carotid artery occlusion in gerbils, the observed reflectance changes were in the same direction (169). Under unilateral ischemia induction, the reflectance trace showed a small decrease (or no change) due to the increase in blood volume through the open artery. However, when complete ischemia was induced (by additionally occluding the other artery), an increase in reflectance was recorded in the preparations. This indicates that a decrease in blood volume may lead to the expected increase in reflectance.
Finally, a decrease in blood volume was induced by saline injection into the ipsilateral common carotid artery or into the brachial artery (Gyulai and Mayevsky, unpublished results). The results showed that the increases in reflectance and fluorescence were similar and varied in the range of 10–20% between various rats or in the same animal.
Changes in mitochondrial NADH and tissue metabolic state
The pioneering work of Chance and Williams in the 1950s, led to the definition of the metabolic state of isolated mitochondria in vitro. The foundations for the use of NADH fluorescence as a marker of mitochondrial activity have been posited in detail by Chance and Williams (56, 57). The left portion of Fig. 6 is a modification of a published table, while the right hand segment demonstrates the responses of NADH fluorescence measured in the brain in vivo under various perturbations. The "resting state" of the mitochondria in vitro was defined as state 4, where NADH was 99% in the reduced form, and ADP was the rate limiting substance. If ADP is added to a suspension of mitochondria, ATP synthesis will be stimulated, O2 consumption will increase, and the rate limit will be determined by the activity of the respiratory chain. During this state 3, or the "active state," the NADH redox state will decrease or become more oxidized (
50%). When the resting mitochondria are deprived of O2, the activity of the mitochondria will stop and NADH will reach its maximum redox state (state 5).
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0) and its maximal level was determined in vitro, it is almost impossible to determine in the intact brain or other organs in vivo. For example, state 2, with a substrate free medium, could not be achieved in vivo since the tissue would die. On the other hand, the maximal level of NADH (state 5) could be monitored in vivo under complete deprivation of O2 by anoxia or complete ischemia.
We used changes in NADH levels monitored in vivo to create a new scale ranging from a maximal definite point to the minimal level recorded in vivo. Details of this approach have been published (152). As shown in Fig. 3, the maximal NADH level is achieved under complete O2 deprivation that can be induced both under in vitro and in vivo conditions. This signifies that this definitive point can be used to determine state 5 in vivo as well. The problem is to determine the metabolic state of a tissue in an in vivo situation. If we adopt the in vitro value of a resting state (state 4), this would signify that the increase in NADH during state 5, induced by anoxia (0% O2), would be only 1%. According to all in vivo studies, this is not the case, and during anoxia the increase in NADH is lager than the decrease under state 4 to 3 transition. Figure 3, right, illustrates that the observed level of NADH increase is indeed larger than the decrease. Therefore, we concluded that, under in vivo conditions, the "resting" metabolic state of the brain is found between states 4 and 3 rather than in state 4 as defined in vitro (152). To determine the maximal and minimal levels of NADH in vivo it is almost impossible to use cyanide or uncoupler (FCCP). Nevertheless, we were able to determine the maximal level by anoxia and the "minimal" level by nonfluorescing uncoupler. We injected the uncoupler pentachlorophenol into the ventricles of the rat's brain while monitoring the NADH responses to anoxia and spreading depression (146). To perform a reliable study with cyanide, the animal would have to die and the results will not be helpful; therefore, we used the anoxia response to measure the maximal level of NADH. Using fiber optic fluorometry, we were able to monitor both anesthetized and awake rats. This figure will be discussed later on in this review. It is important to note that most of the published data on NADH monitoring, have been accumulated in brain studies. Therefore, we will present our data mainly relating to the brain, though results on other organs will be presented as well. Table 1 lists studies published by various investigators as well as our publications. The papers are classified according to the organ monitored and the type of perturbation used. This table does not include rarely studied types of organs or perturbations. Such studies are cited individually in the text.
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ANOXIA AND HYPOXIA.
The responses to hypoxia and anoxia are very similar; therefore, they will be discussed together. According to the definition of Chance and Williams (56, 57), a shift toward state 5 involves an increase in NADH proportional to a decrease in O2 supply.
It is assumed that the response of NADH fluorescence to hypoxia or anoxia, induced in vivo, should be very similar to the response of isolated mitochondria. As shown in Fig. 4B, when the blood-free brain was exposed to N2, the fluorescence showed a clear increase-decrease cycle depending on the availability of O2. The reflectance trace was not affected at all. In autoregulated blood-perfused organs, it is expected that the lack of O2 will trigger compensation mechanisms that may lead to an increase in the blood flow and volume, or a decrease in thereflectancesignal. We tested, in the same rats, the response to anoxia of the normoxic blood-perfused brain. The results are shown in Fig. 4A. Indeed, reflectance exhibited a large decrease due to the increase in blood volume (vasodilatation of brain vessels). Figure 4, C and D, presents the responses to anoxia measured via 2 mm and 1 mm light guides. A small variation can be seen in the reflectance response between the two light guides.
This mechanism is not active in all tissues and therefore different responses of the reflectance signal may be recorded in different tissues. Figure 5 shows the responses of a puppy dog brain to graded hypoxia (A–C) as well as to brain anoxia (D). As it is shown, the changes in the corrected fluorescence signals (CF), which represent the NADH redox state, were inversely correlated to the decrease in FiO2 levels (from 6% to 0% O2).
Figure 6 presents the responses of four rat body organs to graded hypoxia and anoxia. The four organs (the brain, liver, kidney, and testis) show very similar responses to the decrease in O2 supply to the rat. Although the four organs were exposed to the same level of O2 supply, the magnitudes of changes were not identical. Nevertheless, the NADH responses to hypoxia and anoxia clearly show that NADH fluorescence is a good indicator of O2 supply to any of the four organs in the body. Table 1 lists the reports about the effects of anoxia or hypoxia on NADH in various organs. The brain studies were done in vivo, whereas the listed reports on other organs also include several in vitro studies. In our experiments, we have used a short anoxic episode (20–30 s) to test the intactness of the tissue. If the NADH response to anoxia was too small, we stopped the experiment, because it indicated that the brain was damaged and NADH was already elevated. Accordingly, the number of responses to anoxic episodes is very large, and only representative references are cited.
ISCHEMIA, OR DECREASED BLOOD FLOW.
Under partial or complete ischemia, blood flow to the monitored organ is decreased and, as a result, O2 delivery is limited or even abolished. The use of ischemia in animal models provides information relevant to critical clinical situations such as brain stroke or heart attack. The primary factor starting the pathological state is the decrease in O2 supply, making the tissue energy balance negative, and preventing the tissue from performing its function. Figure 7 illustrates the effects of ischemia and anoxia on the NADH level in the brain of an anesthetized gerbil. The measurements of NADH in the cerebral hemispheres were correlated to the brain electrical activity (ECoG; electrocorticogram). To test and compare the measurements done in the two hemispheres, we exposed the gerbil to short-term anoxia. As shown, the two responses are very similar and correlate to the depression of the ECoG signal measured in the two hemispheres.
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Figure 8 presents the responses of a dog heart to lack of O2, monitored in vivo by a fiber optic fluorometer-reflectometer. In parts A and B, the dog was exposed to a short (Fig. 8A) or a longer anoxic episode. In the two anoxic episodes, the NADH was elevated significantly. In the second anoxia, the heart went into fibrillation (Fig. 8B) and the dog died. In Fig. 8C, the coronary artery occlusion led to a very large increase in NADH while the change in the reflectance signal was very small. The changes in blood flow measured by a laser Doppler flowmeter were typical for an ischemic event, as expected. Publications on the effects of ischemia on NADH fluorescence in various organs are listed in Table 1. In all animals or organs used, the NADH was elevated in proportion to the level of ischemia.
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It is well documented that providing animals or man with elevated oxygenation will terminate in the development of "oxygen toxicity." The time needed for the development of this toxic event is inversely proportional to the level of oxygenation, namely the higher the PO2, the shorter the time.
On the other hand, providing more O2 may be beneficial in conditions such as carbon monoxide toxicity, body oxygenation pathology (heart or lung problems), and severe trauma. Therefore, it became necessary to understand the relationship between the level of oxygenation and the function of the mitochondria in vivo.
In the mid-1960s, Chance and collaborators (31, 39, 40) developed the experimental setup that enabled the exposure of various types of mitochondria as well as of the entire small animal in the hyperbaric chamber. They showed that the NADH of the brain, liver, and kidney became oxidized under hyperbaric oxygenation, and this effect was correlated to a decrease in pyridine nucleotides measured by biochemical analysis of fixed tissue.
After the introduction of the light guide-based fluorometry, we were able to expose the awake brain to hyperbaric conditions. A clear decrease in NADH (oxidation) was recorded during the shift from 21% to 100% O2, as well as during compression of up to 10 atmospheres 100% O2 (150, 152, 153, 167, 177). A similar oxidation was found upon CO2 addition to the gas mixture (94–99% O2) (149). We also found a correlation between the elevated brain PO2 and the oxidation of NADH in awake rats (151). The oxidation of NADH was also recorded under normobaric hyperoxia (113). Furthermore, we tested the effects of hyperbaric oxygenation on carbon monoxide intoxication (212) or cyanide exposure (235).
CHANGES IN INSPIRED CO2 AND CO.
The involvement of CO2 in tissue energy metabolism has been studied by several investigators, using higher (hypercapnia) or lower (hypocapnia) CO2 levels in the gas mixture. In 1968, Granholm et al. (90) showed that hyperventilation of a rat led to an increase in NADH probably due to a decrease in cerebral blood flow. Using the monkey brain, Sundt and Anderson (231) and Sundt et al. (233) tested the effects of hyper- and hypocapnia on NADH responses in normoxic and ischemic tissue. No change in NADH was found in the normoxic brain, whereas in the ischemic brain, the change in PCO2 did not affect the elevated NADH. They found a decrease in NADH during seizures, however, nonconcomitant with CO2 changes (236). Under hypocapnia, there was an increase in brain NADH due to a decrease in cerebral blood flow (18, 205). Hypercapnia abolished the oscillation in brain NADH (52). The same group found that hypercapnia did not affect the NADH oxidation level induced by electrical stimulation of the cortex (92, 128, 129). The effects of PCO2 were tested in other organs apart from the brain. Sonn et al. (227) found that dog hypopnea, which induced hypercapnia, changed the redox state of NADH depending on the heart rate.
Exposure of intact animals to CO mimics human CO intoxication requiring an optimal therapeutic strategy (241). Dora et al. (68) were able to change the NADH redox state when CO was applied topically to the brain. Our group showed in detail the influence of various CO levels on the pathophysiology of the brain in vivo (173, 186, 188, 212). When CO levels were low, the NADH remained the same; whereas >3,000 ppm, CO led to an increase in NADH due to the development of hypoxia. In all these studies, we correlated the changes in NADH to other physiological parameters monitored from the same brain area. The negative effects of CO on brain NADH responses to cortical spreading depression were reversed by exposing the rats to hyperbaric oxygenation (212).
Responses to energy consumption changes
As shown by Chance and Williams (57, 58), the activation of the mitochondria by increased ADP is coupled with oxidation of NADH (decreased NADH levels) and is known as the state 4 to state 3 transition in isolated mitochondria. Most of the investigations in this field of tissue activation were made on neuronal tissue in vivo. However, studies of other organs, such as the heart or skeletal muscle, were conducted as well. The demand for energy (ATP) by various tissues is dependent on the specific tasks of each organ or tissue. Nevertheless, the stimulation of mitochondrial function is common in all tissues in the body. We will describe the effects of tissue activation on NADH fluorescence under normoxic conditions as well as during limitation of O2 supply in the tissue (hypoxia, ischemia).
Brain activation.
DIRECT CORTICAL STIMULATION.
Direct cortical stimulation of the cerebral tissue is the less drastic event affecting the energy requirement of the tissue, compared with epileptic activity or cortical spreading depression. Rosenthal and Jobsis correlated the effects of direct cortical stimulation on the fluorescence of the intact cerebral cortex (213). A small but significant oxidation of NADH was recorded depending on the stimulation parameters. The same results were obtained when trains of stimuli were applied to the hippocampus of cats (140). A coupling between extra cellular K+ and the decrease in NADH fluorescence has been documented. This type of study has been later repeated by other investigators who found the same basic pattern of responses. Namely, in the normoxic brain, NADH became oxidized under stimulation (128–130, 139, 141, 143, 226). LaManna et al. (135) found that intensive direct cortical stimulation of the hypoxic cerebral cortex resulted in NADH elevation instead of the typical oxidation (decrease).
BRAIN ACTIVATION BY EPILEPTIC ACTIVITY.
The first detailed report on the influence of epileptiform activity on brain cerebral cortex mitochondrial NADH redox state was published by Jobsis et al. in 1971 (110). With the use of anesthetized cats exposed to an epileptogenic drug (Metrazol or Strychnine), a marked expected oxidation of NADH was recorded. Synchronization of the electrical activity increased the demand for energy to restore ionic homeostasis disturbed by epileptic activity. They had proven that brain hypoxia or anoxia did not develop during the epileptic activity and was not the reason for the termination of bursting. A year later, the same group found similar responses under exposure of cat hippocampus to penicillin (191). During seizures, the decrease in NADH was due to its oxidation and not because of a decrease in NAD+ reduction. The connection between the concentration of K+ and the decrease in mitochondrial NADH was shown in cat hippocampus (192). The effect of seizures on mitochondrial NADH was investigated later on by other groups, mainly in cat models (66, 69, 100, 131, 143, 220, 226, 236, 253, 254). Vern et al. (253) showed that epileptic activity, induced in hypotensive cats, caused an increase in NADH instead of a decrease.
In 1975, we published our first paper on mitochondrial responses to epileptic activity measured in nonanesthetized rats (159). A clear oxidation of NADH was found, even when Metrazol was applied epidurally. Under hypoxia induction (by 10%, 7.5%, and 5% O2), the typical oxidation of NADH during epileptic activity took place, but the curve shape was modified. In most of our studies, we found that the response to epileptic activity was followed by a wave of cortical spreading depression (CSD) (148, 150, 152). We found a similar coupling of NADH oxidation with CSD development, when epileptic activity was developed due to O2 toxicity induced by exposing the rat to hyperbaric oxygenation (151, 153, 167, 177).
Figure 10 presents the progression of seizure activity in a special strain of gerbils, developing epileptic effects as a result of monotonic noise or other factors (168). As seen, the exposure of the awake gerbil to noise resulted in the development of epileptic activity followed by a CSD wave. The coupling between the two pathological events is clear, and manifested by the electrical activity (ECoG) as well as extracellular K+ levels and NADH redox state. During the epileptic stage, extra cellular potassium increased and started to recover to the baseline, but then a larger elevation was recorded. There is a clear correlation between the various parameters during epilepsy and CSD. The decrease in NADH was smaller during the first stage, followed by an oxidation cycle typical for CSD.
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Since our group has published the majority of papers dealing with mitochondrial NADH and CSD, we will start the discussion by describing papers published by others. One of the leading groups, headed by Frans Jobsis, published several studies showing the effects of CSD on brain energy metabolism (135, 136, 143, 215, 226, 234). Several works have been published by Chance and collaborators (96, 97), in addition to those done in collaboration with Mayevsky. Dora's group published two papers in this field (69, 129). Rosenthal and Martel (214) showed that ischemia affected the response to CSD, namely, instead of NADH oxidation, an increase in NADH was recorded (see also Fig. 9, B and C). Recently, Hashimoto et al. (98) used fluorescence imaging to characterize NADH changes in CSD.
Our group has published over 30 papers (see Table 1) on the relationship between mitochondrial NADH redox state and CSD. Also, we reported, for the first time, the development of CSD in the brain of a comatose patient (162): the details will be discussed further on. Typical responses to CSD monitored in the brain of an awake rat are shown in Fig. 9. In Fig. 9A, it is possible to see the correlation between the depression of electrical activity (ECoG) and the oxidation (decrease) of NADH, in triplicate. Figure 9, B and C, shows a comparison between the responses to CSD in a normoxic and partially ischemic brain. As it can be seen, the typical "oxidation cycle" of NADH was replaced in the ischemic brain by a "reduction cycle" due to a decrease in O2 compensation capabilities. This interrelation between the responses to cortical spreading depression and the metabolic state of the brain has been studied in detail by our group (see Table 1).
Activation of body organs. The effects of tissue activity on mitochondrial NADH were investigated in the late 1950s. As shown in Fig. 6, the addition of ADP to resting isolated mitochondria (state 4) may lead to an increase in O2 consumption and oxidation of NADH.
In 1959 Chance and Jobsis (41) applied the newly developed NADH fluorometric method to frog sartorius muscle in vitro and found a decrease in NADH due to a series of twitches leading to an increased ATP breakdown into ADP. Chance (28) applied the same approach to monitor the toad sartorius muscle in vivo and found a clear NADH oxidation depending on the rate of stimulation. Systemic injection of epinephrine into the stimulated muscle induced an increase in NADH. Subjecting the highly stimulated muscle to partial hypoxia, induced a further increase in NADH redox state. Chance's studies of the muscle and NADH were summarized in 1966 (31).
A few years ago, Pal et al. (199) monitored NADH fluorescence in cats subjected to sympathetic trunk stimulation. They found that high stimulation frequency caused a large decrease in capillary blood volume and an increase in NADH levels.
As shown in Brain activation, activation of the brain by either one of the three methods, leads to oxidation of NADH due to the state 4 to state 3 transition of the mitochondria. The prerequisite for such responses is a normal blood flow that has the capacity to increase O2 supply to cope with an increased demand.
Activation of the heart may lead to the development of a more complicated situation, since the blood supply will be affected by changes in the muscle contractility. Using dog heart in vivo, Kedem et al. (114) showed that an increase in the heart rate above 150 beats/min leads to an increase in NADH levels, even when the blood flow is enhanced. These results indicated that the autoregulation of blood flow was insufficient to compensate for the extra O2 needed. The same group tested the effect of inotropic agents on the NADH level in the dog heart in vivo (1). A marked oxidation of NADH was recorded after infusion of norepinephrine or ouabain, concomitantly with the developed increase in tension. The same group published several more studies, in which they monitored NADH in the heart exposed to other physiological and pathophysiological conditions (2, 76). When the heart rate was elevated from 150 to 280 beats/min, the NADH was elevated but the relative levels of NADH were significantly lower after hypopnea (227) or after injection of nitroprusside (76).
Under ischemia induction (230), the NADH became elevated and the local contractile force decreased compared with the simultaneously measured nonischemic region in the left ventricle. An increase in the heart rate in this model, attenuated the typical increase in blood flow and NADH in the nonischemic region. In the ischemic area, a very small reaction was recorded, indicating that some responsiveness to inotropic simulation was retained.
Effects of pharmacological agents.
The effects of various agents on mitochondrial NADH were studied in different organs also under in vivo conditions. Naturally, the in vivo brain was targeted by the most pharmacological agents. The effects of anesthesia or other respiratory chain inhibitors were mainly studied in vitro; however, in vivo experiments were conducted as well. In a pioneering work in 1962, Chance et al. (36) succeeded in showing the different responses of the brain and kidney to Amytal and norepinephrine. Because of inhibition of Site I by Amytal injection, the brain and the kidney showed a clear increase in NADH. After injection of norepnephrine, the brain NADH became oxidized while the kidney NADH was elevated to the level of anoxia. In 1967, Chance showed that in the activated brain (stimulated by Metrazol), the addition of Amytal blocked the epileptic activity and NADH increased in a dose-dependent manner (32). Dora and collaborators (71) significantly contributed to these studies. They tested the influence of phenoxybenzamine on the blood volume and NADH in hypotensive and hypertensive cats. Later on, Dora et al. (72) studied the effects of topical administration of norepinephrine and acetylcholine on blood volume and NADH in the cats' brains. The effects of the adrenergic -blocker on NADH and blood volume were reported (73). The effects of adenosine (127) and other drugs were described by the same group (67). Rex et al. (208) reported on pharmacological manipulations of brain NADH. The effects of other drugs on NADH were studied by Urbanics et al. (249), Vern et al. (255), and Anderson and Meyer (6). Our group published many papers describing the action of various drugs, including anesthetics, on brain NADH in various brain models (176, 182, 258).
The effects of pharmacological agents on NADH redox state in various organs were published as well. Kedem et al. researched the influence of various inotropic agents (1) as well as nitroprusside (2), nitroglycerin (76), and propranolol (86).
Osbakken and collaborators (194, 195) also monitored NADH under various drug exposures. Baron et al. (17) described the effects of lidocaine on NADH, during ischemia in the dog heart. The effects of blood substitute emulsion on NADH in the kidney were reported (260). The influence of radioprotective chemicals on NADH in rat tissue was described in the 1960s (103). The action of various drugs (e.g., the uncoupler Amytal) was studied in the liver exposed to hyperbaric oxygenation (31, 40).
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MONITORING HUMAN BODY ORGANS |
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TOPABSTRACTSPECTROSCOPIC MONITORING OF...SCIENTIFIC BACKGROUND AND...FACTORS AFFECTING NADH...NADH UNRELATED FACTORSMONITORING HUMAN BODY ORGANSMONITORING NADH AND THE...SUMMARY AND CONCLUSIONSREFERENCES |
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The next step was taken by Barlow et al. (16), who expanded this technique to monitor the heart and the brain. Using a different type of fluorometer, Van Buren et al. showed a decrease in NADH (oxidation) due to cortical stimulation in epileptic patients (251). In 1979, Fein and Jobsis (81) studied the changes in brain energetics in patients undergoing superficial temporal arterial-middle cerebral artery microanastomosis. Fein and Olinger (82, 83) monitored patients after transient ischemic attacks. The brain of these patients, who had undergone an extracranial-intracranial bypass, was stimulated, and changes in NADH were recorded.
The laser-based fluorimeter developed by Renault (207) was used to monitor NADH redox state in the heart muscle during pharmacological treatments (207), as well as in skeletal muscle (91). Attempts to apply NADH fluorometry in clinical practice (reported in a dozen short publications) did not lead to the development of a proper medical device applicable on a daily basis.
In 1990, our team started developing a unique multiparametric monitoring system that included the measurement of NADH fluorescence, using a light guide-based device. This system was initially applied to monitor neurosurgical patients undergoing brain surgery or those treated in the intensive care unit. In the first paper on the subject (published in 1991), we showed the feasibility of our approach. After a transient short occlusion of one common carotid artery, the increase in NADH was correlated to a decrease in cerebral blood flow (164). It took another 5 years to restart organized clinical testing of our monitoring system. In the second half of the 1990s (162, 172), we were able to monitor the brain of comatose patients in the intensive care unit and describe the development of spreading depression-like responses, including marked NADH reactions. A modified multiparametric monitoring system was applied for NADH measurement during neurosurgical procedures in the operating room (173). Later on, we attempted to apply our monitoring system to human kidney during a transplantation procedure. We measured the transplanted kidney cortex immediately after the surgical procedures (178). An apparent oxidation of NADH, immediately after reopening the renal artery, was correlated to an increase in microcirculatory blood flow.
Recordings from a head-injured patient (Fig. 11, A and B) demonstrate the importance of the multiparametric monitoring approach. The responses to spreading depression SD (Fig. 11A) were typical for the first five SD cycles, recorded 4.5 h after the beginning of the monitoring period. The responses shown in Fig. 11B, however, were typical for repetitive 35–40 cycles developed every 20–30 min later on in the same patient.
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The observations of human spreading depression using the multiparametric approach (85) and the various responses to it suggest that the basic mechanisms of brain pathophysiological processes may be similar in both the animal and human brain. It is well documented that during the SD wave, which basically involves a propagation of depolarization through the entire hemisphere, a large increase in O2 consumption occurs due to the stimulation of the ion pumps (88). This increased O2 demand is compensated by a large increase in cerebral blood flow.
When SD is induced under relatively normal conditions, there is a complete coupling between the increase in O2 consumption and the large increase in CBF (Fig. 11A). On the other hand, under limited O2 supply (in ischemia or hypoxia), the responses of the brain to SD are altered. The combination of the NADH probe with the CBF probe revealed that the reversed NADH response (an increase instead of a decrease) is due to a limited CBF compensation as indicated by the initial decreased CBF response (Fig. 11B). Such important information may help initiate appropriate intervention procedures to avoid additional deterioration. In the past 10 years, we have attempted to develop a commercial device that could be applied in daily medical practice. In 1999, the first tissue spectroscope was developed and received US Food and Drug Administration clearance in 2000. The device enabled the monitoring of NADH fluorescence and tissue reflectance, in combination with a laser Doppler flowmeter for tissue blood flow measurements. This device was tested in neurosurgical patients in the operating room. The details of the device were published in a few articles (see, for example, Ref. 170). In the past 2 years, an upgraded device has been developed to include another parameter in addition to the original three parameters of the tissue spectroscope. This new device, named the CritiView, could be used to monitor any tissue in the body (155).
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MONITORING NADH AND THE MULTIPARAMETRIC APPROACH |
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In subsequent studies, when we added the monitoring of CBF (laser Doppler flowmetry) to the multiparametric system, we became able to complete the explanation of the SRI event (164). A partial ischemia model in the gerbil revealed that after the initial decrease in blood flow and increase in NADH, the secondary recorded event was very similar to SRI. During the SRI, CBF decreased to very low levels and NADH was further elevated, at the same time as the DC potential and extracellular K+ accumulated as typical for a depolarization event. We concluded that under various pathological brain conditions, depolarization may lead to severe vasoconstriction (decrease in blood volume) due to the accumulated potassium. In this case, an artifact will be recorded in NADH measurements due to the SRI. This detailed example illustrates the advantages of monitoring other physiological parameters in addition to NADH fluorescence. In the next few sections, we will present, in chronological order, the various parameters that have been added to NADH measurements. In all these studies, NADH is the most important parameter, while the inclusion of other parameters improves our ability to explain pathophysiological processes.
NADH and electrical activity
The first attempt to combine NADH and electrical measurements was made by Chance and Schoener in 1962 (50). They showed the time relationship between the increase in NADH due to anoxia or hypoxia, and the disappearance of electrical activity (ECoG) in rat cerebral cortex. The same type of correlation was reported later by Jobsis et al. (110) for epileptic activity, and by Rosenthal and Somjen (163) and Mayevsky and Chance (157) for CSD. The accumulated results have made it clear that under limited energy or O2 supply, NADH becomes elevated in the brain, while the spontaneous ECoG activity is depressed. The ECoG begins to decelerate when NADH reaches 70%-80% of its maximal increase upon death (157, 159) or decapitation (160, 259). The recovery of ECoG after anoxia is completed much later than NADH oxidation, suggesting that energy availability is a prerequisite condition but not the only condition needed for a complete ECoG recovery. Depression of the ECoG is also recorded when the brain is exposed to depolarization due to CSD; however, it is not caused by a lack of O2. Similar correlations between NADH and ECoG were described in cat cerebral cortex exposed to seizures and hemorrhagic hypotension (100).
NADH, ECoG, DC potential, and extracellular K+
In 1974, we correlated the responses of NADH to CSD, with changes in ECoG, extracellular K+ (measured by microelectrodes) and DC steady potential (184). Similar results were published a year later by Lothman et al. (143) and subsequently by other investigators (references not cited).
NADH and respiratory chain components
Since the activities of various respiratory chain components are strongly coupled, the tissue respiratory rate can be better evaluated by monitoring several such components. Very few attempts have been made to correlate NADH responses in vivo, with other components of the respiratory chain. The main reason for this was the stronger interference of blood with Fp or cytochrome oxidase measurements, compared with NADH. The effects of hypotension and anoxia on NADH and cytochrome aa3, were measured in the brain in vivo (99). LaManna et al. showed the effects of Ethanol on brain NADH and cytochrome aa3 in rats and cats (137). Therefore, almost all correlations between Fp and NADH were studied in blood-free organs (49). In 1976, we presented preliminary results indicating that in certain morphological areas of the brain, containing less blood vessels, a good correlation is recorded between NADH and Fp responses to anoxia in vivo (146). The only practical way to measure these two signals together was to freeze the tissue and then analyze the two parameters in the frozen state (168, 183). Another approach to correlating NADH and Fp redox state was suggested by Paddle et al. (198). They used a NADH/Fp scanning fluorometer to monitor the muscle (198) or rat diaphragm (197). A few papers have been published on the use of flying spot fluorometer to monitor the two fluorescent signals in the brain and other organs (35). Most of the data published in this field have been acquired in vitro (33, 49) or in blood-free organs such as the liver (218).
Monitoring NADH, ionic, hemodynamic, and electrical activities
The multiparametric monitoring approach was expanded by Dora et al. (74) by the addition of the PO2 parameter to NADH, reflectance, extracellular K+, DC steady potential, and ECoG. We showed in the gerbil brain that anesthesia affected the NADH, PO2, and ECoG (169).
To improve the multiparametric probe, it was necessary to include the various sensors in a single holder that could be used on a daily basis without too many technical difficulties. In the new probe, all sensors touched the brain surface (after removal of the dura mater without penetration into the tissue itself) (169). Using the same principles, we were able to develop a new generation of multiprobe assemblies that have been used in our laboratory during the past 25 years (Figs. 10 and 11). Here, several relevant publications are cited (85, 150–152, 166). The next significant step was the embedding of a laser Doppler flowmeter sensor into the multiprobe assembly to monitor the continuous microcirculatory blood flow, in addition to NADH, Reflectance, extracellular K+ and Ca2+, and ECoG and DC potential (164). In 1996, we included in the multiprobe assembly a sensor to monitor intracranial pressure (ICP). This device was applied to human brain (162) and rat models (14).
The in vivo multiparametric monitoring approach has been also applied to other organs in the body. Kedem's group supplemented NADH measurements of dog heart, with the monitoring of tissue blood flow by heat clearance and of isometric contractile tension (2). Our group used the multiparametric monitoring system to study the function of rat kidney in vivo (3). The influence of ischemia on NADH and microcirculatory blood flow was studied in rat liver in vivo (15). The correlation between NADH and other parameters, such as microcirculatory blood flow and/or 31P-NMR, was monitored in the beating heart in vivo (194) and in the brain in vivo (180). Presently, due to technological developments, it is possible to integrate the monitoring of NADH with any other type of monitoring systems, either in animal studies or various clinical applications. As mentioned earlier, a unique multiparametric monitoring system based on light-emitting diodes and laser diodes, is available (155). The system is connected to the tissue by a flexible bundle of excitation and emission fibers, and provides real time information on NADH redox state, microcirculatory blood flow, tissue reflectance and HbO2.
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TOPABSTRACTSPECTROSCOPIC MONITORING OF...SCIENTIFIC BACKGROUND AND...FACTORS AFFECTING NADH...NADH UNRELATED FACTORSMONITORING HUMAN BODY ORGANSMONITORING NADH AND THE...SUMMARY AND CONCLUSIONSREFERENCES |
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REFERENCES |
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TOPABSTRACTSPECTROSCOPIC MONITORING OF...SCIENTIFIC BACKGROUND AND...FACTORS AFFECTING NADH...NADH UNRELATED FACTORSMONITORING HUMAN BODY ORGANSMONITORING NADH AND THE...SUMMARY AND CONCLUSIONSREFERENCES |
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 A. Besserer, G. Becard, A. Jauneau, C. Roux, and N. Sejalon-Delmas GR24, a Synthetic Analog of Strigolactones, Stimulates the Mitosis and Growth of the Arbuscular Mycorrhizal Fungus Gigaspora rosea by Boosting Its Energy Metabolism Plant Physiology, September 1, 2008; 148(1): 402 - 413. [Abstract] [Full Text] [PDF]
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 T. L. Clanton Hypoxia-induced reactive oxygen species formation in skeletal muscle J Appl Physiol, June 1, 2007; 102(6): 2379 - 2388. [Abstract] [Full Text] [PDF]
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