Mitochondrial Permeability Transition, Cell Death and Neurodegeneration.

Neurodegenerative diseases are chronic conditions occurring when neurons die in specific brain regions that lead to loss of movement or cognitive functions. Despite the progress in understanding the mechanisms of this pathology, currently no cure exists to treat these types of diseases: for some of them the only help is alleviating the associated symptoms. Mitochondrial dysfunction has been shown to be involved in the pathogenesis of most the neurodegenerative disorders. The fast and transient permeability of mitochondria (the mitochondrial permeability transition, mPT) has been shown to be an initial step in the mechanism of apoptotic and necrotic cell death, which acts as a regulator of tissue regeneration for postmitotic neurons as it leads to the irreparable loss of cells and cell function. In this study, we review the role of the mitochondrial permeability transition in neuronal death in major neurodegenerative diseases, covering the inductors of mPTP opening in neurons, including the major ones-free radicals and calcium-and we discuss perspectives and difficulties in the development of a neuroprotective strategy based on the inhibition of mPTP in neurodegenerative disorders.

Detection of NADH and NADPH levels in vivo identifies shift of glucose metabolism in cancer to energy production.

Profound changes in the metabolism of cancer cells have been known for almost 100 years, and many aspects of these changes have continued to be actively studied and discussed. Differences in the results of various studies can be explained by the diversity of tumours, which have differing processes of energy metabolism, and by limitations in the methods used. Here, using fluorescence lifetime needle optical biopsy in a hepatocellular carcinoma (HCC) mouse model and patients with HCC, we measured reduced nicotinamide adenine dinucleotide (NADH) and reduced nicotinamide adenine dinucleotide phosphate (NADPH) in control liver, and in HCC tumours and their adjacent regions. We found that NADH level (mostly responsible for energy metabolism) is increased in tumours but also in adjacent regions of the same liver. NADPH level is significantly decreased in the tumours of patients but increased in the HCC mouse model. However, in the ex vivo tumour slices of mouse HCC, reactive oxygen species production and glutathione level (both dependent on NADPH) were significantly suppressed. Thus, glucose-dependent NADH and NADPH production in tumours changed but with a more pronounced shift to energy production (NADH), rather than NADPH synthesis for redox balance.

High levels of FAD autofluorescence indicate pathology preceding cell death.

Flavin adenine dinucleotide (FAD) autofluorescence from cells reports on the enzymatic activity which involves FAD as a cofactor. Most of the cellular FAD fluorescence comes from complex II of the electron transport chain in mitochondria and can be assessed with inhibitor analysis. The intensity of FAD autofluorescence is not homogeneous and vary between cells in tissue and in cell culture types. Using primary co-culture of neurons and astrocytes, and human skin fibroblasts we have found that very high FAD autofluorescence is a result of an overactivation of the mitochondrial complex II from ETC and from the activity of monoamine oxidases. Cells with high FAD autofluorescence were mostly intact and were not co-labelled with indicators for necrosis or apoptosis. However, cells with high FAD fluorescence showed activation of apoptosis and necrosis within 24 h after initial measurements. Thus, high level of FAD autofluorescence is an indicator of cell pathology and reveals an upcoming apoptosis and necrosis.

Digital diaphanoscopy of maxillary sinus pathologies supported by machine learning.

Maxillary sinus pathologies remain among the most common ENT diseases requiring timely diagnosis for successful treatment. Standard ENT inspection approaches indicate low sensitivity in detecting maxillary sinus pathologies. In this paper, we report on capabilities of digital diaphanoscopy combined with machine learning tools in the detection of such pathologies. We provide a comparative analysis of two machine learning approaches applied to digital diapahnoscopy data, namely, convolutional neural networks and linear discriminant analysis. The sensitivity and specificity values obtained for both employed approaches exceed the reported accuracy indicators for traditional screening diagnosis methods (such as nasal endoscopy or ultrasound), suggesting the prospects of their usage for screening maxillary sinuses alterations. The analysis of the obtained values showed that the linear discriminant analysis, being a simpler approach as compared to neural networks, allows one to detect the maxillary sinus pathologies with the sensitivity and specificity of 0.88 and 0.98, respectively.

Wireless Dynamic Light Scattering Sensors Detect Microvascular Changes Associated With Ageing and Diabetes.

This article presents clinical results of wireless portable dynamic light scattering sensors that implement laser Doppler flowmetry signal processing. It has been verified that the technology can detect microvascular changes associated with diabetes and ageing in volunteers. Studies were conducted primarily on wrist skin. Wavelet continuous spectrum calculation was used to analyse the obtained time series of blood perfusion recordings with respect to the main physiological frequency ranges of vasomotions. In patients with type 2 diabetes, the area under the continuous wavelet spectrum in the endothelial, neurogenic, myogenic, and cardio frequency ranges showed significant diagnostic value for the identification of microvascular changes. Aside from spectral analysis, autocorrelation parameters were also calculated for microcirculatory blood flow oscillations. The groups of elderly volunteers and patients with type 2 diabetes, in comparison with the control group of younger healthy volunteers, showed a statistically significant decrease of the normalised autocorrelation function in time scales up to 10 s. A set of identified parameters was used to test machine learning algorithms to classify the studied groups of young controls, elderly controls, and diabetic patients. Our conclusion describes and discusses the classification metrics that were found to be most effective.

Assessment of Blood Microcirculation Changes after COVID-19 Using Wearable Laser Doppler Flowmetry.

The present work is focused on the study of changes in microcirculation parameters in patients who have undergone COVID-19 by means of wearable laser Doppler flowmetry (LDF) devices. The microcirculatory system is known to play a key role in the pathogenesis of COVID-19, and its disorders manifest themselves long after the patient has recovered. In the present work, microcirculatory changes were studied in dynamics on one patient for 10 days before his disease and 26 days after his recovery, and data from the group of patients undergoing rehabilitation after COVID-19 were compared with the data from a control group. A system consisting of several wearable laser Doppler flowmetry analysers was used for the studies. The patients were found to have reduced cutaneous perfusion and changes in the amplitude-frequency pattern of the LDF signal. The obtained data confirm that microcirculatory bed dysfunction is present in patients for a long period after the recovery from COVID-19.

HPRT1 Deficiency Induces Alteration of Mitochondrial Energy Metabolism in the Brain.

Alterations in function of hypoxanthine guanine phosphoribosyl transferase (HPRT), one of the major enzymes involved in purine nucleotide exchange, lead to overproduction of uric acid and produce various symptoms of Lesch-Nyhan syndrome (LNS). One of the hallmarks of LNS is maximal expression of HPRT in the central nervous system with the highest activity of this enzyme in the midbrain and basal ganglia. However, the nature of neurological symptoms has yet to be clarified in details. Here, we studied whether HPRT1 deficiency changes mitochondrial energy metabolism and redox balance in murine neurons from the cortex and midbrain. We found that HPRT1 deficiency inhibits complex I-dependent mitochondrial respiration resulting in increased levels of mitochondrial NADH, reduction of the mitochondrial membrane potential, and increased rate of reactive oxygen species (ROS) production in mitochondria and cytosol. However, increased ROS production did not induce oxidative stress and did not decrease the level of endogenous antioxidant glutathione (GSH). Thus, disruption of mitochondrial energy metabolism but not oxidative stress could play a role of potential trigger of brain pathology in LNS.

Laser-induced singlet oxygen selectively triggers oscillatory mitochondrial permeability transition and apoptosis in melanoma cell lines.

Singlet oxygen (1O2) is an electronically excited state of triplet oxygen which is less stable than molecular oxygen in the electronic ground state and produced by photochemical, thermal, chemical, or enzymatic activation of O2. Although the role of singlet oxygen in biology and medicine was intensively studied with photosensitisers, using of these compounds is limited due to toxicity and lack of selectivity. We generated singlet oxygen in the skin fibroblasts and melanoma cell lines by 1267 nm laser irradiation. It did not induce production of superoxide anion, hydrogen peroxide or activation of lipid peroxidation in these cells confirming high selectivity of 1267 nm laser to singlet oxygen. 1O2 did not change mitochondrial membrane potential (ΔΨm) in skin fibroblasts but induced fluctuation in ΔΨm and complete mitochondrial depolarisation due to opening permeability transition pore in B16 melanoma cells. 1267 nm irradiation did not change the percentage of fibroblasts with necrosis but significantly increased the number of B16 melanoma cells with apoptosis. Thus, singlet oxygen can induce apoptosis in cancer B16 melanoma cells by opening of mitochondrial permeability transition pore (PTP) but not in control fibroblasts.

Fluorescence lifetime needle optical biopsy discriminates hepatocellular carcinoma.

This work presents results of in vivo and in situ measurements of hepatocellular carcinoma by a developed optical biopsy system. Here, we describe the technical details of the implementation of fluorescence lifetime and diffuse reflectance measurements by the system, equipped with an original needle optical probe, compatible with the 17.5G biopsy needle standard. The fluorescence lifetime measurements observed by the setup were verified in fresh solutions of NADH and FAD++, and then applied in a murine model for the characterisation of inoculated hepatocellular carcinoma (HCC) and adjacent liver tissue. The technique, applied in vivo and in situ and supplemented by measurements of blood oxygen saturation, made it possible to reveal statistically significant transformation in the set of measured parameters linked with the cellular pools of NADH and NADPH. In the animal model, we demonstrate that the characteristic changes in registered fluorescent parameters can be used to reliably distinguish the HCC tissue, liver tissue in the control, and the metabolically changed liver tissues of animals with the developed HCC tumour. For further transition to clinical applications, the optical biopsy system was tested during the routing procedure of the PNB in humans with suspected HCC. The comparison of the data from murine and human HCC tissues suggests that the tested animal model is generally representative in the sense of the registered fluorescence lifetime parameters, while statistically significant differences between their absolute values can still be observed.

Optical Diagnostics of the Maxillary Sinuses by Digital Diaphanoscopy Technology.

The work is devoted to the development of a scientific and technical basis for instrument implementation of a digital diaphanoscopy technology for the diagnosis of maxillary sinus inflammatory diseases taking into account the anatomical features of patients (differences in skin structure, skull bone thickness, and sinus size), the optical properties of exercised tissues, and the age and gender characteristics of patients. The technology is based on visualization and analysis of scattering patterns of low-intensity radiation as it passes through the maxillary sinuses. The article presents the experimental data obtained using the digital diaphanoscopy method and the results of numerical simulation of the optical radiation passage through the study area. The experimental setup has been modernized through the installation of a a device for controlling the LED applicator brightness. The approach proposed may have considerable promise for creating diagnostic criteria for various pathological changes and can be used to assess the differences in the optical and anatomical features of males and females.

Adrenaline induces calcium signal in astrocytes and vasoconstriction via activation of monoamine oxidase.

Adrenaline or epinephrine is a hormone playing an important role in physiology. It is produced de-novo in the brain in very small amounts compared to other catecholamines, including noradrenaline. Although the effects of adrenaline on neurons have been extensively studied, much less is known about the action of this hormone on astrocytes. Here, we studied the effects of adrenaline on astrocytes in primary co-culture of neurons and astrocytes. Application of adrenaline induced calcium signal in both neurons and astrocytes, but only in neurons this effect was dependent on α- and ß-receptor antagonists. The effects of adrenaline on astrocytes were less dependent on adrenoreceptors: the antagonist carvedilol had only moderate effect on the calcium signal and the agonist of adrenoreceptors methoxamine induced a signal only in small proportion of the cells. We found that adrenaline in astrocytes activates phospholipase C and subsequent release of calcium from the endoplasmic reticulum. Calcium signal in astrocytes is initiated by the metabolism of adrenaline by the monoamine oxidase (MAO), which activates reactive oxygen species production and induces lipid peroxidation. Inhibitor of MAO selegiline inhibited both adrenaline-induced calcium signal in astrocytes and the vasoconstriction that indicates an important role for monoamine oxidase in adrenaline-induced signalling and function.

Interaction of Oxidative Stress and Misfolded Proteins in the Mechanism of Neurodegeneration.

Aggregation of the misfolded proteins ß-amyloid, tau, huntingtin, and α-synuclein is one of the most important steps in the pathology underlying a wide spectrum of neurodegenerative disorders, including the two most common ones-Alzheimer's and Parkinson's disease. Activity and toxicity of these proteins depends on the stage and form of aggregates. Excessive production of free radicals, including reactive oxygen species which lead to oxidative stress, is proven to be involved in the mechanism of pathology in most of neurodegenerative disorders. Both reactive oxygen species and misfolded proteins play a physiological role in the brain, and only deregulation in redox state and aggregation of the proteins leads to pathology. Here, we review the role of misfolded proteins in the activation of ROS production from various sources in neurons and glia. We discuss if free radicals can influence structural changes of the key toxic intermediates and describe the putative mechanisms by which oxidative stress and oligomers may cause neuronal death.

Multimodal Optical Diagnostics of the Microhaemodynamics in Upper and Lower Limbs.

The introduction of optical non-invasive diagnostic methods into clinical practice can substantially advance in the detection of early microcirculatory disorders in patients with different diseases. This paper is devoted to the development and application of the optical non-invasive diagnostic approach for the detection and evaluation of the severity of microcirculatory and metabolic disorders in rheumatic diseases and diabetes mellitus. The proposed methods include the joint use of laser Doppler flowmetry, absorption spectroscopy and fluorescence spectroscopy in combination with functional tests. This technique showed the high diagnostic importance for the detection of disturbances in peripheral microhaemodynamics. These methods have been successfully tested as additional diagnostic techniques in the field of rheumatology and endocrinology. The sensitivity and specificity of the proposed diagnostic procedures have been evaluated.

Optical probe pressure effects on cutaneous blood flow.

The variation of blood flow characteristics caused by the probe pressure during noninvasive studies is of particular interest within the context of fundamental and applied research. It has been shown previously that the weak local pressure induces vasodilation, whereas the increased pressure is able to stop the blood flow in the compressed area, as well as to significantly change optical signals.The blood flow oscillations measured by laser Doppler flowmetry (LDF) characterize the functional state of the microvascular system and can be used for noninvasive diagnostics of its abnormality. This study was intended to identify the patterns of the relationship between the oscillating components of blood flow registered by the LDF method under different levels of pressure applied to an optical fiber probe.For this purpose, we have developed an original optical probe capable of regulating the applied pressure. The developed protocol included six sequential records of the blood perfusion at a pressure within the 0 to 200 mmHg range with unloading at the last stage.Using wavelet analyses, we traced the variation of energy of oscillations for these records in five frequency bands associated with different vascular tone regulation mechanisms. Six young volunteers of the same age (three males and three females) were included in this preliminary study and the protocol was repeated five times in each volunteer. Accordingly, 30 LDF records were available for the analyses. As expected, the LDF signal increases at weak pressure (30 mmHg) and decreases at increased pressure. The statistically stable amplification of endothelial associated blood flow oscillations under the 90 mmHg pressure allowed us to put forward a hypothesis that the endothelial activity increases. The possible causes of this phenomenon are discussed.

Multimodal optical measurement for study of lower limb tissue viability in patients with diabetes mellitus.

According to the International Diabetes Federation, the challenge of early stage diagnosis and treatment effectiveness monitoring in diabetes is currently one of the highest priorities in modern healthcare. The potential of combined measurements of skin fluorescence and blood perfusion by the laser Doppler flowmetry method in diagnostics of low limb diabetes complications was evaluated. Using Monte Carlo probabilistic modeling, the diagnostic volume and depth of the diagnosis were evaluated. The experimental study involved 76 patients with type 2 diabetes mellitus. These patients were divided into two groups depending on the degree of complications. The control group consisted of 48 healthy volunteers. The local thermal stimulation was selected as a stimulus on the blood microcirculation system. The experimental studies have shown that diabetic patients have elevated values of normalized fluorescence amplitudes, as well as a lower perfusion response to local heating. In the group of people with diabetes with trophic ulcers, these parameters also significantly differ from the control and diabetes only groups. Thus, the intensity of skin fluorescence and level of tissue blood perfusion can act as markers for various degrees of complications from the beginning of diabetes to the formation of trophic ulcers.

Combined use of laser Doppler flowmetry and skin thermometry for functional diagnostics of intradermal finger vessels.

We introduce a noninvasive diagnostic approach for functional monitoring of blood microflows in capillaries and thermoregulatory vessels within the skin. The measuring system is based on the combined use of laser Doppler flowmetry and skin contact thermometry. The obtained results suggest that monitoring of blood microcirculation during the occlusion, performed in conjunction with the skin temperature measurements in the thermally stabilized medium, has a great potential for quantitative assessment of angiospatic dysfunctions of the peripheral blood vessels. The indices of blood flow reserve and temperature response were measured and used as the primarily parameters of the functional diagnostics of the peripheral vessels of skin. Utilizing these parameters, a simple phenomenological model has been suggested to identify patients with angiospastic violations in the vascular system.

Computational model of bladder tissue based on its measured optical properties.

Urinary bladder diseases are a common problem throughout the world and often difficult to accurately diagnose. Furthermore, they pose a heavy financial burden on health services. Urinary bladder tissue from male pigs was spectrophotometrically measured and the resulting data used to calculate the absorption, transmission, and reflectance parameters, along with the derived coefficients of scattering and absorption. These were employed to create a "generic" computational bladder model based on optical properties, simulating the propagation of photons through the tissue at different wavelengths. Using the Monte-Carlo method and fluorescence spectra of UV and blue excited wavelength, diagnostically important biomarkers were modeled. Additionally, the multifunctional noninvasive diagnostics system "LAKK-M" was used to gather fluorescence data to further provide essential comparisons. The ultimate goal of the study was to successfully simulate the effects of varying excited radiation wavelengths on bladder tissue to determine the effectiveness of photonics diagnostic devices. With increased accuracy, this model could be used to reliably aid in differentiating healthy and pathological tissues within the bladder and potentially other hollow organs.

Individual variability analysis of fluorescence parameters measured in skin with different levels of nutritive blood flow.

Fluorescence spectroscopy has recently become more common in clinical medicine. However, there are still many unresolved issues related to the methodology and implementation of instruments with this technology. In this study, we aimed to assess individual variability of fluorescence parameters of endogenous markers (NADH, FAD, etc.) measured by fluorescent spectroscopy (FS) in situ and to analyse the factors that lead to a significant scatter of results. Most studied fluorophores have an acceptable scatter of values (mostly up to 30%) for diagnostic purposes. Here we provide evidence that the level of blood volume in tissue impacts FS data with a significant inverse correlation. The distribution function of the fluorescence intensity and the fluorescent contrast coefficient values are a function of the normal distribution for most of the studied fluorophores and the redox ratio. The effects of various physiological (different content of skin melanin) and technical (characteristics of optical filters) factors on the measurement results were additionally studied. The data on the variability of the measurement results in FS should be considered when interpreting the diagnostic parameters, as well as when developing new algorithms for data processing and FS devices.

Substantiation of medical and technical requirements for noninvasive spectrophotometric diagnostic devices.

A scientific approach to the formulation of medical and technical requirements (MTRs) for noninvasive spectrophotometric diagnostic devices using optical technologies such as laser Doppler flowmetry and absorption spectroscopy is proposed. The theoretical modeling framework, metrological certification, and testing of these devices are still in the early stages of development. The theoretical estimation of the received signal levels for wavelengths between 514 and 940 nm is highly dependent on the blood volume level in the subject tissue. The proposed approach allows, in particular, the calculation of technical and metrological performance constraints of the instruments, such as the ranges of the sensitivity and power-related signal-to-noise ratios for different spectral channels and different biomedical (biochemical and physiological) parameters. Substantiation of specialized MTRs for the noninvasive spectrophotometric diagnostic devices can enable them to develop to the level of standardized measurement techniques.