[Possibilities of multiphoton microscopy for the diagnosis of the vulvar lichen sclerosus]. / Vozmozhnosti mul'tifotonnoi mikroskopii dlya diagnostiki skleroticheskogo likhena vul'vy.

BACKGROUND: Vulvar lichen sclerosus (VLS) is a chronic and recurrent dermatosis of an inflammatory nature with severe focal atrophy of the skin. Connective tissue changes are polymorphic and are still not taken into account in histological diagnostics due to the difficulty of interpreting routine histological methods. In this work, we use multiphoton microscopy (MPM) as a new imaging technique that provides detailed information about the organization of collagen fibers in the dermis based on a non-linear second harmonic generation (SHG) process. OBJECTIVE: To determine the degree of connective tissue damage in lichen sclerosus using standard histological techniques and to reveal the diagnostic capabilities of multiphoton microscopy. MATERIAL AND METHODS: We studied 42 biopsies with a histopathological diagnosis of VLS and 10 biopsies of normal vulvar skin. Histological, histochemical and immunohistochemical evaluation was used in comparison with MPM data. Quantitative analysis included the determination of the thickness, length of collagen fibers and the average intensity of the SHG signal. RESULTS: A comprehensive study of the skin showed 4 groups of changes that can be regarded as the degree of the dermis damage: initial, mild, moderate, severe. The affected area at the initial and mild degree has subtle changes, however, it is reliably identified by quantitative analysis of the SHG signal. So, the initial degree is characterized by thin (1.3-1.8 µm) long (56-69 µm) collagen fibers, with a moderate degree, the fibers are thickened (3.4-4.3 µm) and fragmented (22-37 µm). The affected area in moderate and severe cases undergoes homogenization, which is associated with the deposition of extremely thin (0.6-0.9 µm) short (16-28 µm) collagen fibers and the expression of type V collagen. CONCLUSION: Multiphoton microscopy in the second harmonic generation mode is a reliable method for identifying collagen fibers in tissues. The study made it possible to identify 4 degrees of the dermis damage in vulvar lichen sclerosus.

Evaluation of the potential pathogenicity of microorganisms associated with urinary calculi.

Due to the prevalence of postoperative complications in the treatment of urolithiasis, the study of the contamination of urinary calculi and the potential pathogenicity of isolated bacteria is of great importance in laboratory diagnostic practice. It has been shown that uropathogenic bacteria are found in the composition of urinary stones in 65±7.1% of cases, mainly representatives of the Enterobacteriaceae and Staphylococcaceae families. Bacteria of the generas Escherichia, Enterococcus, Staphylococcus were most frequently detected. The analysis of biofilm activity and antibiotic resistance in 50 uropathogenic strains was carried out. It was shown that all the studied strains were resistant to at least two tested drugs, and the average value of the multiple resistance index was 0.51. When cultured on nutrient agar with Congo red, it was shown that more than half of the tested strains have high biofilm activity and about 80% potential for biofilm formation. The greatest biofilm activity was observed in bacteria of the generas Escherichia, Klebsiella, Enterobacter, Staphylococcus.

Controlled Fragmentation of Urinary Stones as a Method of Preventing Inflammatory Infections in the Treatment of Urolithiasis (Experience in Successful Clinical Use).

The introduction of technologically advanced methods of lithotripsy into medical practice changes the nature of postoperative complications. Among them, the main complications are inflammatory infections. This largely determines the search for new, improved methods of stone fragmentation avoiding small stone fragments and dissemination of the pelvicalyceal system of the kidney with stone-associated infection. The authors have developed a method for controlled stone fragmentation using a continuous-wave diode laser with a hot-spot effect at the optical fiber end. The aim of the study was to evaluate the efficacy of controlled urinary stone fragmentation using a continuous-wave diode laser with a highly heated distal end of the optical fiber light guide as a method of preventing inflammatory infections in clinical practice. MATERIALS AND METHODS: We analyzed 1666 case histories of urolithiasis patients who underwent percutaneous nephrolithotripsy/ nephrolithoextraction and contact ureterolithotripsy/ureteroextraction, we also performed a prospective analysis of complications based on the Clavien-Dindo classification in 90 patients who underwent fine fragmentation of stones with various lithotripters: ultrasonic, pneumatic, and holmium laser. The method of controlled stone fragmentation by a diode laser with a hot-spot effect was tested on postoperative samples of 26 renal calculi. For the first time in clinical practice, this method was tested in the bladder cavity (n=10). RESULTS: In the percutaneous nephrolithotripsy group, postoperative infectious and inflammatory complications occurred in 34.1% of cases, in the percutaneous nephrolithoextraction group - in 24.6%, in the contact ureterolithotripsy group - in 7.8%, in the ureterolithoextraction group - in 2.5%. The analysis made it possible to identify factors promoting the development of infectious and inflammatory complications. For the first time in clinical practice, there were successfully performed ten operations of stone fragmentation using a continuous-wave diode laser with a hot-spot effect. Controlled coarse fragmentation of stones providing the possibility to reduce the number of infectious and inflammatory complications was performed in the bladder as a model for testing the method. CONCLUSION: The method of laser-induced controlled coarse fragmentation of stones with a hot-spot effect, developed and tested in clinical practice, is promising for the prevention of infectious and inflammatory complications in patients with potentially infected stones since their fine fragmentation and, consequently, spread of stone-associated toxins and microflora within the urinary system is avoided.

Multiphoton Microscopy and Mass Spectrometry for Revealing Metabolic Heterogeneity of Hepatocytes in vivo.

The aim of the investigation was to study the possibility of revealing the heterogeneity of normal liver hepatocytes in terms of metabolic status using the modern methods of multiphoton microscopy and mass spectrometry. Materials and Methods: Heterogeneity of hepatocytes in terms of total metabolic activity was assessed using multiphoton microscopy based on the autofluorescence intensity of intracellular cofactors NAD(P)H and FAD. Hepatocyte heterogeneity in terms of intensity of intracellular metabolic processes was determined using the fluorescence lifetime imaging (FLIM) method based on the data about fluorescence lifetime contributions of various forms of NAD(P)H. The method of time-of-flight secondary ion mass spectrometry (TоF-SIMS) was used to study the lipid and amino acid composition of hepatocytes. Results: It has been revealed using multiphoton microscopy that hepatocytes are heterogeneous in terms of general metabolic activity. Using FLIM, it was established that the heterogeneity degree was high in terms of intensity of oxidative phosphorylation, glycolysis, and synthetic processes (lipogenesis, nucleic acid synthesis, and the pentose phosphate pathway). The TоF-SIMS method revealed the presence of hepatocyte heterogeneity in terms of amino acid and lipid composition, which points to various intensities of synthetic processes in individual hepatocytes. Moreover, differences in the content of PO3 ions were revealed. The results of ToF-SIMS study correlate with the data obtained by multiphoton microscopy and FLIM, confirming the revealed heterogeneity of hepatocytes in terms of general metabolic activity and intensity of intercellular metabolic processes. Conclusion: The latest methods of fluorescence bioimaging and mass spectrometry proved to be effective in revealing hepatocyte heterogeneity in terms of metabolic status. The presence of heterogeneity should be taken into account in studying the liver tissue under various conditions with the application of fluorescence bioimaging methods.

Accurate early prediction of tumour response to PDT using optical coherence angiography.

Prediction of tumour treatment response may play a crucial role in therapy selection and optimization of its delivery parameters. Here we use optical coherence angiography (OCA) as a minimally-invasive, label-free, real-time bioimaging method to visualize normal and pathological perfused vessels and monitor treatment response following vascular-targeted photodynamic therapy (PDT). Preclinical results are reported in a convenient experimental model (CT-26 colon tumour inoculated in murine ear), enabling controlled PDT and post-treatment OCA monitoring. To accurately predict long-term treatment outcome, a robust and simple microvascular metric is proposed. It is based on perfused vessels density (PVD) at t = 24 hours post PDT, calculated for both tumour and peri-tumour regions. Histological validation in the examined experimental cohort (n = 31 animals) enabled further insight into the excellent predictive power of the derived early-response OCA microvascular metric. The results underscore the key role of peri-tumour microvasculature in determining the long-term PDT response.

[The role of pharmacological preconditioning in renal ischemic and reperfusion injury].

Renal ischemic and reperfusion injury resulting in acute renal failure is a multidisciplinary problem at the junction of pathophysiology, transplantology, urology, nephrology, cardiac surgery and pharmacology. One of renal protection strategies is using the phenomenon of preconditioning. Preconditioning is one of the ways to adopt a tissue to repeated short-term effects of damaging factors to induce an enhanced tolerance to the long period of hypoxia and/or ischemia. There are multiple cellular and molecular mechanisms of the renal protective effects of preconditioning stimuli, but the key effectors and signaling molecules are ATP-dependent potassium channels, nitric oxide synthase, nitric oxide, and mitochondrial pore. Contradictory data on the protective effect of ischemic preconditioning allow searching for approaches to pharmacological correction of ischemic and reperfusion injuries. The article provides data on possible ways of using erythropoietin, darbepoetin and phosphodiesterase 5 inhibitors.

Photodynamic therapy monitoring with optical coherence angiography.

Photodynamic therapy (PDT) is a promising modern approach for cancer therapy with low normal tissue toxicity. This study was focused on a vascular-targeting Chlorine E6 mediated PDT. A new angiographic imaging approach known as M-mode-like optical coherence angiography (MML-OCA) was able to sensitively detect PDT-induced microvascular alterations in the mouse ear tumour model CT26. Histological analysis showed that the main mechanisms of vascular PDT was thrombosis of blood vessels and hemorrhage, which agrees with angiographic imaging by MML-OCA. Relationship between MML-OCA-detected early microvascular damage post PDT (within 24 hours) and tumour regression/regrowth was confirmed by histology. The advantages of MML-OCA such as direct image acquisition, fast processing, robust and affordable system opto-electronics, and label-free high contrast 3D visualization of the microvasculature suggest attractive possibilities of this method in practical clinical monitoring of cancer therapies with microvascular involvement.

In Vitro Effect of Laser-Induced Hydrodynamics on Cancer Cells.

We studied the effect of laser-induced hydrodynamic on viability of Colo-26 murine colon carcinoma cells in vitro. Laser-induced hydrodynamics was generated by a laser (λ=1.56 µ, power 3 W, 5 min exposure); to this end, the fiber end was submersed into a buffer above the cell monolayer. It was found that laser-induced hydrodynamics destructed the monolayer at standoff distances of between the working end of the laser fiber to cell monolayer of 1 and 5 mm and triggers apoptotic and necrotic death in remaining cells at a distance of 4 mm from the emitter.

[Laser hyperthermia of tumors with the use of golden nanoparticles under the control of optical coherent tomography and acoustothermometry].

The local laser hyperthermia of an experimental tumor RShM-5 of mice with the use of golden plasmin resonance nanoparticles has been carried out. The accumulation of particles in the tumor was controlled by the in vivo noninvasive method of optical coherent tomography. Using this method, the time of the maximum content of nanoparticles in the tumor was determined to be 5 h after the intravenous administration during which the laser hyperthermia was performed. The control of the tumor temperature during the hyperthermia seance showed that the application of nanoparticles provides an effective temperature elevation inside the node and a more targeted heating. The local laser hyperthermia with nanoparticles induced the inhibition of the tumor growth from day 5 to day 7 after the seance with a maximum value of 56%.

[Pathogenetic therapy of trigeminal neuralgia]. / K patogeneticheskoi terapii nevralgii troinichnogo nerva.

The authors describe a clinical case of severe neuralgia of the third branch of the trigeminal nerve, in whose etiology and pathogenesis a role was played by allergic vasomotor rhinosinusopathy, general allergization of the body, and recurrent herpetic infection. The patient was treated by carbamazepine, tranquilizers, prednisolone, antihistamine and diuretic agents, calcium drugs, bonaphthon, vitamin B1, essentiale, acupuncture, local hydrocortisone phonophoresis and laser therapy. Such treatment made it possible to effectively remove the neuralgic painful syndrome. The given case demonstrates that the painful syndrome is due to several pathological systems having different pathophysiological and neurochemical organization, demanding a differentiated individual approach and providing evidence for the necessity of carrying out the etiological and pathogenetic therapy.