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.

Deconvolution of the fluorescence spectra measured through a needle probe to assess the functional state of the liver.

OBJECTIVES: Currently, one of the most pressing issues for surgeons in the treatment of obstructive jaundice is the ability to assess the functional state of the liver and to detect and determine the degree of liver failure in a timely manner with simple and objective techniques. In this regard, the use of fluorescence spectroscopy method can be considered as one of the ways to improve the informativity of existing diagnostic algorithms in clinical practice and to introduce new diagnostic tools. Thus, the aim of the work was to study in vivo the functional state of liver parenchyma by the method of fluorescence spectroscopy implemented through a needle probe and assess the contribution of the main tissue fluorophores to reveal new diagnostic criteria. MATERIALS AND METHODS: We compared data from 20 patients diagnosed with obstructive jaundice and 11 patients without this syndrome. Measurements were performed using a fluorescence spectroscopy method at excitation wavelengths of 365 and 450 nm. Data were collected using a 1 mm fiber optic needle probe. The analysis was based on the comparison of the results of deconvolution with the combinations of Gaussian curves reflecting the contribution of the pure fluorophores in the liver tissues. RESULTS: The results showed a statistically significant increase in the contribution of curves reflecting NAD(P)H fluorescence, bilirubin, and flavins in the group of patients with obstructive jaundice. This and the calculated redox ratio values indicated that the energy metabolism of the hepatocytes may have shifted to glycolysis due to hypoxia. An increase in vitamin A fluorescence was also observed. It may also serve as a marker of liver damage, indicating impaired vitamin A mobilization from the liver due to cholestasis. CONCLUSIONS: The results obtained reflect changes associated with shifts in the content of the main fluorophores characterizing hepatocyte dysfunction caused by accumulation of bilirubin and bile acids and after disturbance of oxygen utilization. The contributions of NAD(P)H, flavins, and bilirubin as well as vitamin A can be used for further studies as promising diagnostic and prognostic markers for the course of liver failure. Further work will include collecting fluorescence spectroscopy data in patients with different clinical effects of obstructive jaundice on postoperative clinical outcome after biliary decompression.

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.

Interaction of Mitochondrial Calcium and ROS in Neurodegeneration.

Neurodegenerative disorders are currently incurable devastating diseases which are characterized by the slow and progressive loss of neurons in specific brain regions. Progress in the investigation of the mechanisms of these disorders helped to identify a number of genes associated with familial forms of these diseases and a number of toxins and risk factors which trigger sporadic and toxic forms of these diseases. Recently, some similarities in the mechanisms of neurodegenerative diseases were identified, including the involvement of mitochondria, oxidative stress, and the abnormality of Ca2+ signaling in neurons and astrocytes. Thus, mitochondria produce reactive oxygen species during metabolism which play a further role in redox signaling, but this may also act as an additional trigger for abnormal mitochondrial calcium handling, resulting in mitochondrial calcium overload. Combinations of these factors can be the trigger of neuronal cell death in some pathologies. Here, we review the latest literature on the crosstalk of reactive oxygen species and Ca2+ in brain mitochondria in physiology and beyond, considering how changes in mitochondrial metabolism or redox signaling can convert this interaction into a pathological event.

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.

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.