NADH Fluorescence Images Research Articles

The use of NADH anisotropy to investigate mitochondrial cristae alignment

Life may be expressed as the flow of electrons, protons, and other ions, resulting in large potential difference. It is also highly photo-sensitive, as a large proportion of the redox capable molecules it relies on are chromophoric. It is thus suggestive that a key organelle in eukaryotes, the mitochondrion, constantly adapt their morphology as part of the homeostatic process. Studying unstained in vivo nano-scale structure in live cells is technically very challenging. One option is to study a central electron carrier in metabolism, reduced nicotinamide adenine dinucleotide (NADH), which is fluorescent and mostly located within mitochondria. Using one and two-photon absorption (340–360 nm and 730 nm, respectively), fluorescence lifetime imaging and anisotropy spectroscopy of NADH in solution and in live cells, we show that mitochondria do indeed appear to be aligned and exhibit high anisotropy (asymmetric directionality). Aqueous solution of NADH showed an anisotropy of ~ 0.20 compared to fluorescein or coumarin of < 0.1 and 0.04 in water respectively and as expected for small organic molecules. The anisotropy of NADH also increased further to 0.30 in the presence of proteins and 0.42 in glycerol (restricted environment) following two-photon excitation, suggesting more ordered structures. Two-photon NADH fluorescence imaging of Michigan Cancer Foundation-7 (MCF7) also showed strong anisotropy of 0.25 to 0.45. NADH has a quantum yield of fluorescence of 2% compared to more than 40% for photoionisation (electron generation), when exposed to light at 360 nm and below. The consequence of such highly ordered and directional NADH patterns with respect to electron ejection upon ultra-violet (UV) excitation could be very informative—especially in relation to ascertaining the extent of quantum effects in biology, including electron and photonic cascade, communication and modulation of effects such as spin and tunnelling.

NADH fluorescence imaging and the histological impact of cortical spreading depolarization during the acute phase of subarachnoid hemorrhage in rats.

OBJECTIVE Although cortical spreading depolarization (CSD) has been observed during the early phase of subarachnoid hemorrhage (SAH) in clinical settings, the pathogenicity of CSD is unclear. The aim of this study is to elucidate the effects of loss of membrane potential on neuronal damage during the acute phase of SAH. METHODS Twenty-four rats were subjected to SAH by the perforation method. The propagation of depolarization in the brain cortex was examined by using electrodes to monitor 2 direct-current (DC) potentials and obtaining NADH (reduced nicotinamide adenine dinucleotide) fluorescence images while exposing the parietal-temporal cortex to ultraviolet light. Cerebral blood flow (CBF) was monitored in the vicinity of the lateral electrode. Twenty-four hours after onset of SAH, histological damage was evaluated at the DC potential recording sites. RESULTS Changes in DC potentials (n = 48 in total) were sorted into 3 types according to the appearance of ischemic depolarization in the entire hemisphere following induction of SAH. In Type 1 changes (n = 21), ischemic depolarization was not observed during a 1-hour observation period. In Type 2 changes (n = 13), the DC potential demonstrated ischemic depolarization on initiation of SAH and recovered 80% from the maximal DC deflection during a 1-hour observation period (33.3 ± 15.8 minutes). In Type 3 changes (n = 14), the DC potential displayed ischemic depolarization and did not recover during a 1-hour observation period. Histological evaluations at DC potential recording sites showed intact tissue at all sites in the Type 1 group, whereas in the Type 2 and Type 3 groups neuronal damage of varying severity was observed depending on the duration of ischemic depolarization. The duration of depolarization that causes injury to 50% of neurons (P50) was estimated to be 22.4 minutes (95% confidence intervals 17.0-30.3 minutes). CSD was observed in 3 rats at 6 sites in the Type 1 group 5.1 ± 2.2 minutes after initiation of SAH. On NADH fluorescence images CSD was initially observed in the anterior cortex; it propagated through the entire hemisphere in the direction of the occipital cortex at a rate of 3 mm/minute, with repolarization in 2.3 ± 1.2 minutes. DC potential recording sites that had undergone CSD were found to have intact tissue 24 hours later. Compared with depolarization that caused 50% neuronal damage, the duration of CSD was too short to cause histological damage. CONCLUSIONS CSD was successfully visualized using NADH fluorescence. It propagated from the anterior to the posterior cortex along with an increase in CBF. The duration of depolarization in CSD (2.3 ± 1.2 minutes) was far shorter than that causing 50% neuronal damage (22.4 minutes) and was not associated with histological damage in the current experimental setting.

Age-related metabolic fatigue during low glucose conditions in rat hippocampus

Previous reports have indicated that with aging, intrinsic brain tissue changes in cellular bioenergetics may hamper the brain's ability to cope with metabolic stress. Therefore, we analyzed the effects of age on neuronal sensitivity to glucose deprivation by monitoring changes in field excitatory postsynaptic potentials (fEPSPs), tissue Po2, and NADH fluorescence imaging in the CA1 region of hippocampal slices obtained from F344 rats (1–2, 3–6, 12–20, and >22 months). Forty minutes of moderate low glucose (2.5 mM) led to approximately 80% decrease of fEPSP amplitudes and NADH decline in all 4 ages that reversed after reintroduction of 10 mM glucose. However, tissue slices from 12 to 20 months and >22-month-old rats were more vulnerable to low glucose: fEPSPs decreased by 50% on average 8 minutes faster compared with younger slices. Tissue oxygen utilization increased after onset of 2.5 mM glucose in all ages of tissue slices, which persisted for 40 minutes in younger tissue slices. But, in older tissue slices the increased oxygen utilization slowly faded and tissue Po2 levels increased toward baseline values after approximately 25 minutes of glucose deprivation. In addition, with age the ability to regenerate NADH after oxidation was diminished. The NAD+/NADH ratio remained relatively oxidized after low glucose, even during recovery. In young slices, glycogen levels were stable throughout the exposure to low glucose. In contrast, with aging utilization of glycogen stores was increased during low glucose, particularly in hippocampal slices from >22 months old rats, indicating both inefficient metabolism and increased demand for glucose. Lactate addition (20 mM) improved oxidative metabolism by directly supplementing the mitochondrial NADH pool and maintained fEPSPs in young as well as aged tissue slices, indicating that inefficient metabolism in the aging tissue can be improved by directly enhancing NADH regeneration.

In VivoNADH Fluorescence Imaging Indicates Effect of Aquaporin-4 Deletion on Oxygen Microdistribution in Cortical Spreading Depression

Using in vivo two-photon imaging, we show that mice deficient in aquaporin-4 (AQP4) display increased fluorescence of nicotinamide adenine dinucleotide (NADH) when subjected to cortical spreading depression. The increased NADH signal, a proxy of tissue hypoxia, was restricted to microwatershed areas remote from the vasculature. Aqp4 deletion had no effects on the hyperemia response, but slowed [K(+)]o recovery. These observations suggest that K(+) uptake is suppressed in Aqp4(-/-) mice as a consequence of decreased oxygen delivery to tissue located furthest away from the vascular source of oxygen, although increased oxygen consumption may also contribute to our observations.

Label-freein vivoimaging of human leukocytes using two-photon excited endogenous fluorescence

We demonstrate that two-photon excited endogenous fluorescence enables label-free morphological and functional imaging of various human blood cells. Specifically, we achieved distinctive morphological contrast to visualize morphology of important leukocytes, such as polymorphonuclear structure of granulocyte and mononuclear feature of agranulocyte, through the employment of the reduced nicotinamide adenine dinucleotide (NADH) fluorescence signals. In addition, NADH fluorescence images clearly reveal the morphological transformation process of neutrophils during disease-causing bacterial infection. Our findings also show that time-resolved NADH fluorescence can be potentially used for functional imaging of the phagocytosis of pathogens by leukocytes (neutrophils) in vivo. In particular, we found that free-to-bound NADH ratios measured in infected neutrophils increased significantly, which is consistent with a previous study that the energy consumed in the phagocytosis of neutrophils is mainly generated through the glycolysis pathway that leads to the accumulation of free NADH. Future work will focus on further developing and applying label-free imaging technology to investigate leukocyte-related diseases and disorders.

Dynamic changes in cortical NADH fluorescence in rat focal ischemia: Evaluation of the effects of hypothermia on propagation of peri-infarct depolarization by temporal and spatial analysis

Suppression of peri-infarct depolarizations (PIDs) is one of the major mechanisms of hypothermic protection against transient focal cerebral ischemia. Previous studies have shown the lack of hypothermic protection against permanent focal ischemia. We hypothesized the lack of hypothermic protection was due to the poor efficacy in suppression of PIDs. To examine the hypothesis, we elucidated the effects of hypothermia on the manner of propagation of PIDs with temporal and spatial resolutions using NADH (reduced nicotinamide adenine dinucleotide) fluorescence images by illuminating the parietal–temporal cortex with ultraviolet light. Spontaneously hypertensive rats ( n = 14) were subjected to permanent focal ischemia by occlusion of the middle cerebral and left common carotid arteries. 2-h hypothermia (30 °C) was initiated before ischemia. Although hypothermia delayed the appearance of PIDs, it did not suppress their appearance. Furthermore, 54% of the PIDs enlarged the high-intensity area of NADH fluorescence in the hypothermia group, similar to the normothermia group (53%). The high-intensity area of NADH fluorescence widened by each PID was larger in the hypothermia group than in the normothermia group. These findings suggest that PIDs even in hypothermia are one of the major factors causing growth of infarction, emphasizing the importance of therapy that targets suppression of PIDs even during hypothermia.

High‐Resolution Intravital NADH Fluorescence Microscopy Allows Measurements of Tissue Bioenergetics in Rat Ileal Mucosa

NADH fluorescence microscopy has been used as an index of the metabolic state of tissue but is associated with various obstacles such as low spatial resolution and quenching effects of blood pigments that prevent reliable monitoring of tissue bioenergetics. The objective of this study was to develop a system to monitor tissue bioenergetics in vivo using NADH fluorescence microscopy in the rat ileal mucosa. Using an inverted microscope with an epifluorescence unit and an intensified charge-coupled device camera, NADH fluorescence images were visualized. Fluorescence intensity was measured of beta-NADH solutions at varying concentration (n = 6) and pH (n = 3) and in ex vivo (n = 6) and in vivo (n = 6) preparations of ileal mucosa of Sprague-Dawley rats anesthetized with isoflurane. Intravital fluorescence microscopy reveals a map of the microcirculation that permits visualization of NADH fluorescence and intercapillary areas. The system was adjusted so a linear relationship between physiological concentrations of beta-NADH and fluorescence was achieved (r(2) = 0.98, p < .0001). Decreasing the pH of the solution had no effect on fluorescence intensity and fluorescence intensity in an anoxic ex vivo ileal segment was similar to that of the in vivo ileum after ischemia. Ischemia also resulted in spatial heterogeneity that was abolished by the addition of a 550-nm LP filter. With this system, intravital NADH fluorescence microscopy provides the high resolution necessary to reliably monitor tissue bioenergetics in the rat ileal mucosa.

Fluctuation analysis of mitochondrial NADH fluorescence signals in confocal and two-photon microscopy images of living cardiac myocytes.

A fluctuation analysis was performed on the reduced nicotine adenine dinucleotide (NADH) fluorescence signal from resting rabbit myocytes using confocal and two-photon microscopy. The purpose of this study was to establish whether any co-ordinated biochemical processes, such as binding, metabolism and inner mitochondrial membrane potential, were contributing to NADH signal fluctuations above background instrument noise. After a basic characterization of the instrument noise, time series of cellular NADH fluorescence images were collected and compared with an internal standard composed of NADH in the bathing medium. The coefficient of variation as a function of mean signal amplitude of cellular NADH fluorescence and bathing media NADH was identical even as a function of temperature. These data suggest that the fluctuations in cellular NADH fluorescence in resting myocytes are dominated by sampling noise of these instruments and not significantly modified by biological processes. Further analysis revealed no significant spatial correlations within the cell, and Fourier analysis revealed no coherent frequency information. These data suggest that the impact of biochemical processes, which might affect cellular NADH fluorescence emission, are either too small in magnitude, occurring in the wrong temporal scale or too highly spatially localized for detection using these standard optical microscopy approaches.

Dynamic changes in cortical NADH fluorescence and direct current potential in rat focal ischemia: relationship between propagation of recurrent depolarization and growth of the ischemic core.

Forty rats were subjected to 3 hours of focal ischemia by occluding the left middle cerebral and left common carotid arteries. The propagation of recurrent depolarization around the ischemic core was analyzed using direct-current potential and NADH (reduced nicotinamide adenine dinucleotide) fluorescence images by irradiating the parietal-temporal cortex with ultraviolet light. Based on histological evaluation at direct-current recording sites, the total time of depolarization causing 50% neuronal injury was estimated to be 18.2 minutes. The sites showing recurrent depolarizations resulted in 23 +/- 29% neuronal injury due to the short depolarization time, whereas the sites showing recurrent depolarizations and eventually persistent depolarization resulted in infarction. The NADH fluorescence images showed that recurrent depolarizations propagated along the margin of the ischemic core. In 85.9% of the recurrent depolarizations, the fluorescence disappeared without leaving any traces and did not affect the area of the ischemic core. However, in 47.5% of the animals, 14.1% of recurrent depolarizations merged with the ischemic core and increased the area by 6 +/- 4 mm(2). These findings suggest that recurrent depolarization increases the severity of neuronal injury but does not cause infarction by itself if persistent depolarization does not follow, and that the area of persistent depolarization is enlarged with 14.1% of recurrent depolarizations.

Pretransplantation assessment of renal viability with NADH fluorimetry

A pathophysiologic feature possibly involved in ischemic injury in transplant kidneys is mitochondrial dysfunction caused by disintegration of oxidative metabolic pathways. Because the ability to synthesize ATP by respiratory activity determines the organ's capacity to recover from ischemic injury, an assessment of respiratory activity may provide information related to graft viability. NADH fluorimetry can be used to monitor kidney cortex metabolism noninvasively. During perfusion with (an)-aerobic perfusate, NADH fluorescence images were recorded. We evaluated the NADH oxidation kinetics of 20 rat kidneys, which were divided over four experimental groups. For six minimally damaged kidneys and six kidneys that had been stored for one hour at 37 degrees C, perfusion was followed by transplantation. We related the kinetic parameters of these kidneys with their post-transplantation function and histology. The transplant function was monitored by serum creatinine and urea levels. Storage of transplant kidneys for one hour at 37 degrees C significantly reduced the post-transplantation function. Isolated perfusion of grafts, however, was not detrimental for renal function. The rate of NADH oxidation decreased with decreasing graft quality, and a good correlation between NADH oxidation kinetics and post-transplantation function was found. A reduction of NADH oxidation rates as a consequence of warm ischemia supports the view that mitochondrial respiratory activity is impaired by ischemic injury. The correlation between NADH oxidation kinetics in perfused grafts and their post-transplantation function indicates that NADH fluorimetry may be useful in predicting the viability of preserved grafts prior to transplantation.

Dynamic changes of NADH fluorescence images and NADH content during spreading depression in the cerebral cortex of gerbils

To elucidate the changes in the mitochondrial redox state during spreading depression (SD), tissue NADH content was measured in 20 anesthetized gerbils by the enzymatic cycling assay in a small cortical region (0.30±0.07 mg) where the direct-current (DC)-potential was measured. Sequential imaging of NADH fluorescence with a CCD camera and continuous monitoring of DC-potential and regional CBF were also performed in another 5 gerbils. Biphasic fluorescence waves propagating at the rate of 3 mm/min were observed using the CCD camera. An initial narrow (1.6±0.4 mm) wave, which showed a modest increase in fluorescence (108±6.4%), was observed simultaneously with the onset of negative DC-deflection. During depolarization, CBF was unchanged and tissue NADH content increased to 25.3±7.9 μmol/kg brain, which was higher than the value in the sham-control (11.0±2.5 μmol/kg brain). At 30 s after the deflection, a subsequent wide (7.0±2.1 mm) wave, which showed a moderate decrease in fluorescence (87.1±5.7%), was observed simultaneously with the increase in CBF and repolarization in DC-potential. Then NADH fluorescence recovered along with normalization of CBF at 152.2±38.6 s after the onset of DC-deflection. Tissue NADH concentration sampled at 120 s after the deflection was 11.6±4.6 μmol/kg brain. Since NADH fluorescence is absorbed by hemoglobin, the initial increase and subsequent decrease in fluorescence seem to have been induced by increases in NADH content and CBF, respectively. These findings indicate that the mitochondrial redox state transiently inclines to the reduction side synchronous to the onset of DC-deflection and that it normalizes within 120 s after deflection.

NADH Videofluorimetry to Monitor the Energy State of Skeletal Musclein Vivo

Background.Reduction of the cellular energy state during ischemia of the limbs is an important determinant for development of necrosis. Since energy conversion in the mitochondria is based on electron transport from NADH to molecular O2, the NADH/NAD+redox couple reflects the mitochondrial redox state and cellular O2requirement. The applicability of NADH videofluorimetry to monitor noninvasively changes in the energy state of intact resting skeletal muscle as a function of oxygenation was investigated in a rat model.Materials and methods.In mechanically ventilated rats (n= 6), NADH fluorescence images of the gracilis muscle were recorded under different oxygenation conditions. Induction of anoxic and ischemic hypoxia were verified by simultaneous measurement of tissue oxygen pressure and afferent blood flow.Results.Anoxic hypoxia and ischemic hypoxia increased the NADH fluorescence intensity by 46.0 ± 15.0 and 30.8 ± 26.4%, respectively. The response time of NADH fluorescence intensity, tissue oxygen pressure, and afferent blood flow was similar during development of anoxic and ischemic hypoxia. Upon reperfusion and reoxygenation, however, NADH fluorescence intensity changed significantly earlier than tissue oxygen pressure as measured by an oxygen needle electrode.Conclusion.These results demonstrate that changes in NADH fluorescence intensity reflect oxygenation changes in intact skeletal musclein vivo.Since NADH videofluorimetry, in contrast to oxygen needle electrode measurements, noninvasively visualizes temporal and regional changes in the energy state of skeletal muscle, this technique has the potential to improve clinical evaluation of ischemia/reperfusion injury and tissue viability.

Weak spots inside the myocardium of a Langendorff rat heart observed by NADH videofluorometry.

Myocardial function is highly dependent on an aerobic metabolism and can only be maintained when the oxygen delivery is more than the oxygen demand. Dysoxia occurs when the demand of oxygen by the heart exceeds that being supplied through the myocardial vasculature. Hypoperfusion of the myocardium causes a marked heterogeneity of well and less well perfused areas. These areas have been shown to be anatomically defined by the properties of the myocardial microvasculature (King et al. 1985, Franzen et al. 1988, Ince et al. 1993, Rakusan et al. 1994, Duling 1994). The properties of these dysoxic areas under different physiological conditions have been most studied by NADH fluorescence imaging techniques (Barlow et al. 1976, Steenbergen et al. 1977, Ince et al. 1993, Vetterlein et al. 1995). The NADH fluorescence technique was introduced by Chance and makes use of the fluorescence properties of mitochondrial NADH for the measurement of mitochondrial energy state of tissue cells in situ (Chance et al. 1976). NADH is situated at the high-energy side of the respiratory chain and during dysoxia accumulates in concentration because less NADH is oxidised to NAD+. Episodes of high cardiac oxygen consumption, as occurs during high work rates, causes reduction of NADH levels (Osbakken 1994, Ashruf et al. 1995). The optical properties of NADH and NAD+ clearly differ. Upon excitation with UV-light (365 nm) NADH, unlike NAD+, fluoresces at the 460 nm light (blue). Under borderline hypoxic conditions imaging the distribution of NADH in the heart surface shows a heterogeneous distribution of well, and less well perfused areas. The less well perfused areas we have termed as cardiac “weak spots” since they are first to become dysoxic when the heart becomes comprised such as occurs during severe tachycardia (Ince et al. 1993) or during severe vasoconstriction as occurs during nitric oxide synthesis inhibition in endotoxemic hearts (Avontuur et al. 1995).KeywordsWeak SpotMitochondrial NADHPerfuse AreaNADH FluorescenceHigh Work RateThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Inhibition of Nitric Oxide Synthesis Causes Myocardial Ischemia in Endotoxemic Rats

Inhibitors of nitric oxide (NO) synthesis have been used in the treatment of septic and endotoxic shock. However, several studies question the beneficial effect of inhibiting NO production in sepsis and endotoxemia. We have investigated the effect of inhibition of NO synthesis after endotoxemia in the isolated perfused rat heart. In hearts from endotoxin-treated animals, coronary flow was elevated 64% and oxygen consumption was elevated 20% compared with control hearts. NADH fluorescence imaging was used as an indicator of regional hypoperfusion. A homogeneous low-surface NADH fluorescence, indicative of adequate tissue perfusion, was observed in both control and endotoxin-treated hearts. The increase in coronary flow and oxygen consumption could only partially be prevented by pretreatment of the animals with dexamethasone. Addition of N omega-nitro-L-arginine (NNLA), an inhibitor of NO synthesis, to the perfusion medium eliminated differences in coronary flow and oxygen consumption between normal and endotoxin-treated hearts. However, NADH surface fluorescence images of endotoxin-treated hearts after NNLA revealed areas of high fluorescence, indicating local ischemia, whereas the control hearts remained without signs of ischemia. The ischemic areas were present at various perfusion pressures and disappeared after the infusion of L-arginine, the natural precursor of NO, or the exogenous NO donor sodium nitroprusside. Methylene blue (MB), an inhibitor of soluble guanylate cyclase, the effector enzyme of NO, also eliminated differences in coronary flow and produced similar areas of local myocardial ischemia in endotoxin-treated hearts but not in control hearts.(ABSTRACT TRUNCATED AT 250 WORDS)

Spreading cortical depression of Leao.

Spreading cortical depression of Leao.