Laser-Induced Fluorescence Spectroscopy in the Diagnosis of Tissue Hypoxia (Review)

Цель обзора — анализ опыта диагностики тканевой гипоксии методом лазер-индуцированной спектроскопии, а также выявление перспективных направлений и потенциальных возможностей данного метода для дальнейшего его применения в экспериментальной и клинической медицине. В обзоре представили данные исследований интенсивности флуоресценции эндогенных молекул-флуорофоров (никотинадениндинуклеотида восстановленного, флавинадениндинуклеотида окисленного) как маркеров ишемического повреждения внутренних органов (мозга, сердца, печени, почек и др.). Рассмотрели принципы метода флуоресцентной лазер-индуцированной спектроскопии in vivo. Уделили внимание историческим аспектам темы. Показали результаты применения метода в экспериментальных и клинических исследованиях тканевой гипоксии и ишемии. Выявили трудности в интерпретации значений интенсивности аутофлуоресцентного сигнала исследуемых молекул. Отметили, что неизвестным остается период сохранения способности ткани к аутофлуоресценции в условиях длительной аноксии, отсутствуют систематизированные представления о влиянии экзогенных и эндогенных факторов на интенсивность аутофлуоресценции. Сделали вывод, что применение метода лазер-индуцированной флуоресцентной спектроскопии с целью диагностики тканевой ишемии является перспективным направлением в экспериментальной и клинической медицине, не исчерпавшим себя, и оставляющим ряд нерешенных вопросов, несмотря на большое количество исследований в данной области.


Ключевые слова: лазер-индуцированная флуоресценция; НАДН; ФАД; гипоксия; эндогенные флуорофоры; тканевая гипоксия; ишемия головного мозга
The aim of review is to discuss the results of studies on diagnosis of tissue hypoxia by laser-induced spectroscopy, as well as to identify promising trends and prospects of this technique for its further application in experimental and clinical medicine.
The review presents the findings of studies of the fluorescence intensity of endogenous fluorophore molecules (reduced nicotinamide adenine dinucleotide, oxidized flavin adenine dinucleotide) as markers of ischemic injury of internal organs (brain, heart, liver, kidneys, etc.).The principles of fluorescence laser-induced spectroscopy in vivo are discussed.The historical aspects of the subject are also covered.The results of the use of the technique in experimental and clinical studies of tissue hypoxia and ischemia are shown.Difficulties in interpreting the intensity values of autofluorescent signal of the studied molecules are revealed.It was noted that the tissue autofluorescence in a long-term anoxia remains unknown, and there are no structured ideas about the impact of exogenous and endogenous factors on autofluorescence intensity.
In conclusion, the use of laser-induced fluorescence spectroscopy to diagnose tissue ischemia is a promising area of experimental and clinical medicine, which still has various unresolved issues, despite a large number of studies in this domain.

Introduction
Tissue resistance to hypoxia and anoxia is a fundamental topic in medicine of a multidisciplinary significance.Oxygen starvation is the cause of tissue metabolism disorders in case of blood loss, blood circulation disorders, traumatic injuries, acute poisoning and other critical conditions [1][2][3].Studying the mechanisms of hypoxia/anoxia as a pathological process and modern methods of its diagnosis attracts the attention of clinicians of any specialty.In-situ diagnosis of tissue hypoxia in experimental models, as well as in clinical medicine, offers a possibility of intraoperative monitoring of organs and tissues, tissue viability determination in circulatory failure after transplantation.Therefore, it is necessary to place more emphasis on methods that allow to diagnose tissue hypoxia in situ in real time.One of such methods is laser-induced autofluorescence spectroscopy.
The aim of this literature review is to examine the available data on diagnosing the tissue hypoxia by laser-induced spectroscopy, as well as to identify the promising directions and prospects of this technique for its further application in experimental and clinical medicine.
Fundamentals of autofluorescence spectroscopy.Autofluorescence spectroscopy is based on the detection of fluorescence intensity of endogenous fluorophore molecules.The phenomenon of autofluorescence consists in emitting quanta of light by molecules excited due to the effects of radiation with a specific long wavelength, when changing from a metastable state with a higher energy level to the basic state with the lowest energy [4,5].
The most important diagnostic endogenous tissue fluorophores are tryptophan, collagen, elastin, reduced nicotinamide adenine dinucleotide (NADH), as well as its phosphate (NADPH), flavins and flavoproteins (FAD, FMN), porphyrins, beta-carotene.Pyridine and flavin nucleotides, which are located to a greater extent in mitochondria and to a lesser extent in cytoplasm, are one of the main sources of cellular fluorescence [6].They participate in a wide range of intracellular processes, such as glycolysis, pentose cycle, Krebs cycle, fatty acid oxidation, mitochondrial respiratory chain, and biosynthetic processes, and can be used as markers of cellular metabolism in normal and pathological processes [7][8][9][10].The NADH coenzyme fluoresces only in the reduced form and the maximum excitation is registered on exposure to radiation with a wavelength of 340 nm, while the quantum fluorescence yield is greater if NADH is bound to the protein.Flavin nucleotides are only capable of fluorescence in oxidized form.The maximum excitation is registered on exposure to radiation with a wavelength of 450 nm [5,7].Based on  [20].
History of the method.Since the discovery of the optical properties of the reduced nicotinamide adenine dinucleotide in 1950, it has been intensively studied as a marker of mitochondrial function.The founder of this area of research, Britton Chance, has proven through his numerous studies, both in vitro and in vivo, that mitochondrial function can be evaluated by the redox state of NADH.A major milestone in the history of coenzyme research was the creation of a two-beam spectrophotometer by B. Chance in 1954 [19].Metabolic characteristics of isolated mitochondria as a function of substrate, oxygen and ADP level were studied for the first time by a spectrophotometry.In 1959, Chance and Legallais developed a differential microfluorimeter for studying coenzymes not only in cell suspensions and ultra-thin tissue samples, but also in animal tissues in vivo.In 1962, the first works by Chance et al. appeared, where monitoring of NADH was done on anesthetized rat organs.
In 1971, Jobsis et al. pioneered this method in the brain of neurosurgical patients with focal epilepsy in vivo.A correlation between NADH and electrocorticography data was found in direct cortical stimulation of the controlled areas.The obtained results were consistent with those of a previous similar experimental study [20].
Due to difficulties in interpreting the tissue autofluorescence, the use of fluorescence spectroscopy has become widespread in medicine only with the introduction of exogenous fluorophores.However, the interest in autofluorescence spectroscopy of tissues in vivo has been demonstrated by a number of studies ranging from the works of B. Chance et al. to modern Russian and foreign research dealing with the role of tissue autofluorescence in malignant neoplasms, neurodegenerative diseases, ischemia and reperfusion [21].
Diagnosing the ischaemic injury of internal organs by laser-induced fluorescence spectroscopy.The cells switch to anaerobic respiration as a result of hypoxia.Changes in the respiratory enzyme chain are initialized at the NADH-dependent site with a short-term increase in functioning, followed by suppression of this mitochondrial complex site.Lack of oxygen leads to the accumulation of reduced nicotinamide coenzyme (NADH), fluorescing in ultraviolet light, which makes it possible to use NADH as a marker of mitochondrial dysfunction caused by hypoxia/anoxia.Autofluorescence fading is observed with continued anoxia [22][23][24].
In the early days of the method implementation in experimental medicine, C. H. Barlow (1977) studied the fluorescence of NADH in myocardium during coronary occlusion and identified its changes after 15 seconds from the moment of coronary artery ligation.In the study by F. H. Tomlinson, brain ischemia was associated with a 150% increase in NADH fluorescence 15 minutes after carotid occlusion [31].
Fitzgerald J. T. et al. showed in their experiment the efficacy of laser autofluorescent imaging in the diagnosis of ischemic and reperfusion damage of the kidneys in vivo and assessment of their viability after a period of renal artery occlusion [32].Tirapelli L. F. et al. successfully used autofluorescence spectroscopy to study the response to longterm ischemia and reperfusion in the experiment, and also found a correlation between the intensity of renal fluorescence in ischemic and reperfusion damage and histological signs of renal, as well as mitochondrial damage [33,34].Raman R. N. et al. suggested using multimodal autofluorescence spectroscopy to predict the post-transplantation kidney function [35].
The study of NAD(F)H and FAD as autofluorescent biomarkers of metabolic disturbances and liver and hepatocyte damage began in the 1950s.The purpose of later studies was to monitor the liver response to ischemia/reperfusion. Subsequently, fluorescent fatty acids, lipopigments, and collagen were characterized as optical biomarkers of liver steatosis and fibrosis [36,37].M. F. Lacour et al. demonstrated a correlation between the redox ratio (NADH/FAD) and other markers of hepatic injury on a 60-minute and 90minute liver ischemia model with and without reperfusion [38].
Croce A. C. et al. studied the autofluorescence properties of bile and concluded that autofluorescence bile spectroscopy can provide additional information to assess changes in the liver metabolic activity balance [39].
Arutyunyan A. V. et al. demonstrated in an experiment the high efficacy of intraoperative use of the laser-induced fluorescence spectroscopy method for diagnostics of pancreas tissue necrosis in situ at acute pancreatitis [40].
Staniszewski K. et al. studied the intensity of NADH and FAD fluorescence of isolated perfused rat lungs after adding rotenon, mitochondrial complex I (NADH-ubiquinone-oxidoreductase) inhibitor; potassium cyanide, complex IV inhibitor (C-oxidase cytochrome), and pentachlorophenol, tissue respiration inhibitor, into the perfusate.Changes in the metabolic state of mitochondria in the lungs affected the intensity of coenzyme fluorescence.The autofluorescence signal decreased after adding blood to the perfusate, but the redox ratio remained unchanged.This study demonstrated the sensitivity of coenzyme fluorescence intensity and redox ratio to changes in redox processes in mitochondria [43].
Diagnosis of cerebral ischemia.The diagnostic significance of brain autofluorescence became apparent from the findings of B. Chance's early studies.The first use of laser-induced fluorescence spectroscopy in vivo in the clinic was carried out on neurosurgical patients for a reason [20].A number of studies have shown a correlation between changes in the fluorescence signals of the reduced NADH and the oxidized FAD and the functional activity of neuronal and glial mitochondria, intracellular concentration of calcium and its transmembrane transport of calcium in mitochondria [44][45][46][47].
Despite the experimentally and clinically proven applicability of the fluorescence intensity indexes of NADH and FAD coenzymes as biomarkers of ischemic brain damage [48][49][50][51][52][53][54][55], the method has some limitations associated with the difficulty of data interpretation.In the in vitro study, Kahraman S. et al. demonstrated differences in the fluorescence mechanisms of pyridine nucleotides in neural and glial cells.Persisting anoxia after the initial increase in NADH fluorescence in neurons was accompanied by its gradual decrease, which was not observed in astrocytes.There is also a need to differentiate the changes in the autofluorescence of NAD(P)H caused by metabolic disorders from the changes induced by the stimulation of local blood flow by the increased lactate production during ischemia [56].
Polesskaya O. et al. developed a technique based on the relationship between the intensity of endogenous fluorescence of NADH and partial oxygen pressure in the tissue, allowing to diagnose the microregional hypoxia of the murine cerebral cortex [57].
In the light of several studies, the NADH and FAD autofluorescence intensity indices serve as markers not only for hypoxic brain damage, but also for neoplastic and neurodegenerative processes.In 2017, Shi L. et al. in experimental studies showed the possibility of using NADH, FAD and DOI:10.15360/1813-9779-2019-6-50-61
Pascu A. et al. have demonstrated that the comparative assessment of autofluorescence ranges of normal and neoplastic tissue helps to identify them.
The results of the experimental in vitro study led the authors to suggest using the fluorescence spectroscopy for the real-time assessment of neoplastic and adjacent normal tissue autofluorescence during surgery in vivo [59].Zhu M. B. et al. supplemented the autofluorescence spectroscopy with optical coherence tomography, which increased the sensitivity of the tumor tissue identification from 86.1 to 95.9% [60].
Ibrahim B. A. et al. showed the effect of temperature on the fluorescence of NADH and FAD coenzymes in the rat cortex.An association between an increase in temperature and a decrease in FAD fluorescence was found [61].
A study by Reinert K. C. et al. showed that autofluorescence of cerebellar flavoproteins is related to inhibition and excitation processes [62].
The study of NADH and FAD coenzymes in ischemic brain damage not only proved the possibility of their use as damage and regeneration markers, but also gave rise to the development of drugs containing pyridine nucleotides which reduce the area of necrosis in the brain, improve the functional recovery, and reduce mortality in experimental animals after ischemic stroke [63].
Diagnosis of myocardial ischemia.Since autofluorescence spectroscopy of NADH and FAD coenzymes helps to evaluate tissue metabolism in real time, the use of this method for intraoperative cardiac monitoring is a promising aspect of clinical and experimental medicine.
Papayan G. et al. based on the results of studies of NADH and FAD coenzymes in ischemiareperfusion of an isolated rat heart recommended this technique for intraoperative monitoring of myocardial metabolism during episodes of anoxia in patients who underwent cardiac surgery under cardiopulmonary bypass [64].
Lagarto J. L. et al. on the Langedorff isolated heart model revealed changes in FAD and NAD(P)H fluorescence associated with the glucose and oxygen depletion.Meanwhile, the coenzyme fluorescence intensity was more sensitive to changes in oxygen content in the perfusate than to changes in glucose level.The potential for using autofluorescence spectroscopy to estimate the content of type I collagen in the post-infarct period was demonstrated [65][66][67].
Despite the hypothetical background and the correlation between autofluorescence and rejection of the cardiac allograft found in experimental studies with rats, study on the use of laser-induced autofluorescence spectroscopy to assess the risk of G Reviews ФАД и триптофана для ранней диагностики болезни Альцгеймера [58].
A number of papers deal with the use of this technique to control the condition of the myocardium in ischemia in cardioprotection.The study of NADH fluorescence changes in relation to the reperfusion period duration and type of cardioplegia conducted by T. Nishioka (1984) was among the earliest in this area.The incomplete recovery of coenzyme fluorescence level was explained by irreversible changes in myocardium [31].Horvath et al. studied the changes of laser-induced NADH fluorescence in the myocardium during sequential occlusion and cold cardioprotection.Immediately after the occlusion, a rapid increase in the fluorescence of NADH was recorded over a period of less than 5 min, followed by a decline over the entire period of ischemia, which lasted for 2 hours.The FAD fluorescence patterns shown in this study was different: within 5 minutes after occlusion, there was a rapid decrease in fluorescence, followed by a slow decrease during 30 minutes, a steady level during 1 hour and a slow rise until the end of the study [69].In a study by Aldakkak et al. (2009), the changes in NADH and FAD levels in myocardial ischemia induced by cardioplegia with hyperkalemic solution and lidocaine were evaluated.Different patterns of NADH changes with and without cardioplegia were demonstrated [70].
Camara A. K. and colleagues studied the cardioprotective effects of sevoflurane, warm reperfusion and hypothermia on the cold ischemia model in isolated guinea pig hearts, measuring metabolism using NADH and FAD levels.Ischemia caused a rapid increase in NADH and a decrease in FAD, which declined in 2 hours.Warm reperfusion resulted in a decrease in NADH and an increase in FAD.Sevoflurane attenuated changes in NADH and FAD and reduced the size of infarction [71].
Changes in the intensity of NADH and FAD autofluorescence in the myocardium during ischemia, reperfusion, in cardiomyopathy and cardioprotection found in a number of studies have made it possible to consider them as markers of myocardial ischemic injury [72][73][74][75][76][77][78][79][80].

Conclusion
The literature review shows that despite extensive research on this topic the interpretation of the auto-fluorescent signal intensity and the determination of the hypoxia/anoxia period are still open issues.Tissue autofluorescence has been studied quite well and explained theoretically only in the case of short-term ischemia.Studies on ischemia continuing for 60-90 minutes are rare, and they have provided no indication of tissue autofluorescence changes in complete lack of oxygen over the period of tissue death.Little attention has been DOI:10.15360/1813-9779-2019-6-50-61Обзоры лорода.При этом интенсивность флуоресценции коферментов была более чувствительна к изменению содержания кислорода в перфузате, чем к изменению уровня глюкозы.Была показана возможность использования метода аутофлуоресцентной спектроскопии для оценки содержания коллагена I типа в постинфарктном периоде [65][66][67].

Acknowledgments
The author would like to express her sincere gratitude to professor A. M. Golubev, MD, PhD, DSci (V. A. Negovsky Scientific Research Institute of General Reanimatology) for his help in writing this review and for his valuable comments.и увеличению ФАД.Севофлюран ослаблял изменения НАДН и ФАД и уменьшал размер инфаркта [71].