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Objective — To investigate the apoptotic activity of Gratiola officinalis L. extract on human tumor cell lines by flow cytofluorometry. Material and Methods — The extract of Gratiola officinalis L. was manufactured via our original methodology. Studies were performed on human tumor cell lines: HeLa – cervical carcinoma, Jurkat – T-cell lymphoblastic leukemia, MCF-7 – breast adenocarcinoma, A549 – lung carcinoma, PC-3 – prostate carcinoma, HCT-116 – colon carcinoma, A498 – renal carcinoma, and SK-BR-3 – human breast carcinoma. Induction of apoptosis was studied after incubating cell lines with Gratiola officinalis L. extract at a concentration of 0.9 mg/mL using the Annexin V-FITC Apoptosis Kit. Caspase-dependent apoptosis was examined on a flow cytometer using anti-caspase-3-FITC (BD) kit on the Jurkat cell line. Morphological studies of HeLa cervical carcinoma cells in the alive and dead test were performed using two stains, acridine orange and propidium iodide, at different concentrations of the extract. The statistical data processing was performed using Microsoft Office Excel software. Results — One day after their exposure to Gratiola officinalis L. extract at a concentration of 0.9 mg/mL, tumor cells were mostly in late apoptosis stage. Cytotoxic activity of Gratiola officinalis L. extract was established for all investigated tumor cell cultures but their sensitivities to the extract were different. Mechanisms of antitumor action of Gratiola officinalis L. extract were identified: we established that the extract induced caspase-dependent apoptosis in tumor cells. Conclusion — The identified mechanisms of apoptotic activity of Gratiola officinalis L. extract confirmed the prospects of bioflavonoids as new-generation antitumor agents.

Optical clearing of the lung tissue aims to make it more transparent to light by minimizing light scattering, thus allowing reconstruction of the three-dimensional structure of the tissue with a much better resolution. This is of great importance for monitoring of viral infection impact on the alveolar structure of the tissue and oxygen transport. Optical clearing agents (OCAs) can provide not only lesser light scattering of tissue components but also may influence the molecular transport function of the alveolar membrane. Air-filled lungs present significant challenges for optical imaging including optical coherence tomography (OCT), confocal and two-photon microscopy, and Raman spectroscopy, because of the large refractive-index mismatch between alveoli walls and the enclosed air-filled region. During OCT imaging, the light is strongly backscattered at each air-tissue interface, such that image reconstruction is typically limited to a single alveolus. At the same time, the filling of these cavities with an OCA, to which water (physiological solution) can also be attributed since its refractive index is much higher than that of air will lead to much better tissue optical transmittance. This review presents general principles and advances in the field of tissue optical clearing (TOC) technology, OCA delivery mechanisms in lung tissue, studies of the impact of microbial and viral infections on tissue response, and antimicrobial and antiviral photodynamic therapies using methylene blue (MB) and indocyanine green (ICG) dyes as photosensitizers.

Objective — To compare the expression of the LC3B autophagy marker before and after treatment with high-intensity focused ultrasound therapy (HIFU) and combined therapies: androgenic deprivation AD + HIFU, AD + radiation therapy in patients with and without recurrent prostate adenocarcinoma. Material and Methods — The objects of our study were biopsy specimens of prostates obtained from 70 patients with prostate adenocarcinoma before and after treatment. The first group consisted of patients without signs of relapse after treatment, as evidenced by a normal blood PSA level, the results of ultrasound procedure and biopsy. The second group included patients who had a recurrence of adenocarcinoma after treatment. Patients received the following cancer treatments: ultrasound ablation (HIFU), combination therapy: androgenic deprivation AD + HIFU, AD + radiation therapy. An immunohistochemical staining in two groups was performed with an anti-LC3B antibody. Results — In the group of patients without relapse, the expression of the autophagy marker sharply decreased in the tumor tissue after treatment, but it increased in the stroma of muscle connective tissue and the vascular wall. In the group of patients with relapse, the expression of anti-LC3B antibody significantly increased in tumor cells, in stroma of muscle connective tissue, and in vascular wall after treatment. In both groups, the magnitude of the autophagy marker expression did not depend on the treatment method. Conclusion — The autophagy is one of the factors contributing to the survival of prostate adenocarcinoma cells after treatment; the increase in autophagic protein expression after treatment in patients with prostate adenocarcinoma can be used to assess the risk of relapse.

Serum samples of healthy rats and rats with inoculated tumors on days 14 and 28 after transplantation of cholangiocarcinoma cells were analyzed using Raman spectroscopy (RS). It was shown that the intensity of the amide 1 band differs in the serum spectra depending on the stage of tumor development and correlates with the protein concentration in the samples. In some animals, there is a significant increase in the blood glucose concentration, leading to glycation of proteins and a change in the shape of the amide 1 band. The appearance of a band at 1670 cm–1 is most pronounced in the spectra of samples on the 28th day of the experiment and may be associated with an increase in the content of β-structural elements in the protein conformation. Using the principal component analysis, we assessed the general differences in the Raman spectra of the blood serum in rats of control groups and rats on the 28th day after tumor transplantation.

The purpose of this study was to investigate the prognostic factors of plasmonic photothermal therapy (PPT) efficiency in rats with transplanted liver cancer on the basis of assessment of the degree of tumor vascularization. Before any exposure, rats with transplanted liver cancer PC-1 were subjected to Doppler ultrasonography to assess the degree of vascularization of transplanted tumors. Half of the animals with the transplanted tumors were withdrawn from the experiment after Doppler ultrasonography to determine the content of vascular factors in blood serum by enzyme immunoassay and in tumor tissue by immunohistochemical method. After three intravenous injections of polyethylene glycol-coated gold nanorods (GNRs) at a dose of 0.4 mq/mL, the transplanted tumors were irradiated percutaneously with infrared laser radiation at a wavelength of 808 nm, followed by thermography of local tumor heating. The animals were withdrawn from the experiment 24 h later, and tumor tissue samples were taken for histological examination. It is found that the accumulation of gold in the tumor tissue and the PPT efficiency at repeated intravenous GNR injections are determined by the presence of formed vasculature in the tumor.

The quercetin content in aqueous EtOH extracts of covering leaves of anthocyanin Purple Saratov diploid variety of Zea mays L. grown under dry conditions reached 39.36 ± 0.41 mg/100 g of dry extract and significantly exceeded the value (26.03 ± 0.36 mg/100 g of dry extract) for maize grown under normal wetting conditions. The large amounts of polyphenolic compounds in anthocyanin Purple Saratov diploid variety showed good prospects for using it as a source of them.

Optical clearing (OC) of adipose tissue has not been studied enough, although it can be promising in medical applications, including surgery and cosmetology, for example, to visualize blood vessels or increase the permeability of tissues to laser beams. The main objective of this work is to develop technology for OC of abdominal adipose tissue in vivo using hyperosmotic optical clearing agents (OCAs). The maximum OC effect (77%) was observed for ex vivo rat adipose tissue samples exposed to OCA on fructose basis for 90 minutes. For in vivo studies, the maximum effect of OC (65%) was observed when using OCA based on diatrizoic acid and dimethylsulfoxide for 120 minutes. Histological analysis showed that in vivo application of OCAs may induce a limited local necrosis of fat cells. The efficiency of OC correlated with local tissue damage through cell necrosis due to accompanied cell lipolysis.

Background and aim — Gratiola officinalis extract has antimicrobial properties, but its wound healing activity has not been described. Material and Methods — The aqueous solution of dry Gratiola officinalis extract was used for treatment of skin wounds with different routes of administration. In the experiment 40 white laboratory male rats with weight of 150±50 grams were subjected to the incised model of flat skin wounds in the interscapular region under the general anesthesia. A morphological study was carried out to assess the effectiveness of the therapy administered. Results — The most effective method of treatment was found to be local application of the extract, which reduced wound healing time by 8 days. Intramuscular and oral administrations reduce healing time by 6 days. Morphological signs of regeneration, which developed under the influence of Gratiola officinalis extract, allowed to estimate the wound healing activity. Clear differentiation and thickness of layers of the newly formed epidermis, a large number of thin-walled vessels and actively proliferating fibroblasts indicate an active regeneration process. Conclusion — This extract may be interesting to specialists in regenerative medicine as a remedy for treatment of wound processes.

Cancer remains one of the leading causes of death in the world. For a number of neoplasms, the efficiency of conventional chemo- and radiation therapies is insufficient because of drug resistance and marked toxicity. Plasmonic photothermal therapy (PPT) using local hyperthermia induced by gold nanoparticles (AuNPs) has recently been extensively explored in tumor treatment. However, despite attractive promises, the current PPT status is limited by laboratory experiments, academic papers, and only a few preclinical studies. Unfortunately, most nanoformulations still share a similar fate: great laboratory promises and fair preclinical trials. This review discusses the current challenges and prospects of plasmonic nanomedicine based on PPT and photodynamic therapy (PDT). We start with consideration of the fundamental principles underlying plasmonic properties of AuNPs to tune their plasmon resonance for the desired NIR-I, NIR-2, and SWIR optical windows. The basic principles for simulation of optical cross-sections and plasmonic heating under CW and pulsed irradiation are discussed. Then, we consider the state-of-the-art methods for wet chemical synthesis of the most popular PPPT AuNPs such as silica/gold nanoshells, Au nanostars, nanorods, and nanocages. The photothermal efficiencies of these nanoparticles are compared, and their applications to current nanomedicine are shortly discussed. In a separate section, we discuss the fabrication of gold and other nanoparticles by the pulsed laser ablation in liquid method. The second part of the review is devoted to our recent experimental results on laser-activated interaction of AuNPs with tumor and healthy tissues and current achievements of other research groups in this application area. The unresolved issues of PPT are the significant accumulation of AuNPs in the organs of the mononuclear phagocyte system, causing potential toxic effects of nanoparticles, and the possibility of tumor recurrence due to the presence of survived tumor cells. The prospective ways of solving these problems are discussed, including developing combined antitumor therapy based on combined PPT and PDT. In the conclusion section, we summarize the most urgent needs of current PPT-based nanomedicine.

We report here a pilot identification of the presence of the lumenized Lyve-1/Prox-1-expressing vessels with distinct walls composed of a single endothelial layer in the unaffected brain and with intraventricular hemorrhages. These lymphatic vessels (LVs) have valves and an undulating shape in the distal region that is the classical characteristic of lymphatic precollectors. Furthermore, we identified Lyve-1/Prox-1-expressing lymphatic elements in the enlarged perivascular spaces. These pioneering results might be a revolutionary step in our understanding of anatomy and physiology of the cerebral lymphatics and stimulate a reassessment of basic assumptions in the aetiology of brain diseases associated with lymphatic dysfunction. The discovery of the cerebral LVs is a crucial step in the development of breakthrough technologies of modulations of lymphatic removing of toxins and blood from the brain.One Sentence SummaryLyve1/Prox1-expressing lymphatic vessels and elements in the unaffected and affected human brain.

Goal — to assess the effect of antenatal hypoxia of various origins on the morphology and reproductive function of the testes of newborn and mature rats in experiment. Material and Methods — In experiments 15 white outbred female rats aged 4 to 10 months with a weight of 200±30 g were used. Laboratory animals were randomly divided into 2 experimental and 1 control groups, 5 females each. The first group underwent normobaric hypoxia throughout pregnancy (21 days). Hypoxia modeling was conducted in accordance with the method of N.N. Karkishchenko (2010). The second group underwent hemic hypoxia during the second and third week of pregnancy, in accordance with the method of L.M. Sosedova (2012). The third (control) group was not exposed to any effect throughout pregnancy. Results — in the testicles of newborn rats of the experimental groups, the decrease of tubule diameter was observed, the increase of stroma area and development of interstitial edema were noted. In the group of hemic hypoxia, a significant decrease in the number of Leydig cells was noted. In the tissues of the testicles of mature rats, who underwent antenatal hypoxia, a decrease of tubule diameter, a significant decrease in the spermatogenesis index and a decrease of spermatogonia number were noted. The developed damage of spermatogenic epithelium in experimental groups of newborns and mature rats was confirmed by marked expression of the apoptosis marker (Bax), weak expression of proliferation markers (Ki-67) and receptor of receptor of fibroblast growth factor (FGFR). Conclusion — in animals with chronic hypoxia of various origins, there is an inhibition of spermatogenesis and a violation of the spermatogenetic function of the testicular seminiferous tubules.

The paper presents the investigation of change of optical properties of the rat transplanted cholangiocarcinoma doped with gold nanorods after laser-induced plasmon-resonant photothermal treatment (PPT). 72, 48 and 24 hours before the experiment the animals were injected with the suspension of gold nanorods intravenously. For irradiation a diode laser with wavelength 808 nm was used. After the irradiation the tumors and surrounding tissues were removed and sliced. The samples studied were: skin, subcutaneous connective tissue, tumor capsule, top, center, and bottom part of tumor. Spectra of total transmittance and diffuse reflectance of the samples were measured in the wavelength range 350-2250 nm. Absorption and reduced scattering coefficients of the tumor tissues were calculated with inverse adding-doubling method. The results of the experiment were compared with result of investigation of the control tissues without gold nanorods injection and PPT. A decrease in the absorption coefficient of tumor layers in the water absorption bands was obtained, which indicated tissue dehydration during PPT. A decrease in the reduced scattering coefficient of tumor layers indicated an increase in the size of scatterers and an increase in their ordering. Changes in the optical parameters of the skin and subcutaneous layer were insignificant, which indicated a weak thermal damage.

Our previous study has revealed that specific ornithosis antigens extracted from Сhlamydia psittaci (CP) could possess some modulatory activity on the growth of malignant solid tumors in Wistar rats. Here, in the same model we studied dose-dependent dynamics of tumor growth under the influence of CP antigen derived from CP AMK-16 strain, in doses ranged from 0.025 mg/kg to 1.25 mg/kg given as a single subcutaneous injection. Velocity of tumor growth has been estimated by t-LASCA technique.

The goal of our research was to assess the vascularization degree of tumor to predict the efficiency of plasmonic photothermal therapy (PPT) and photodynamic therapy (PDT) in tumor-bearing rats. Before any treatment, 3D Doppler ultrasound imaging was used for assessment of the vascularization degree of transplanted rat cholangiocarcinoma. For PPT, the gold nanorods with aspect ratio of 4:1, functionalized with thiolated polyethylene glycol, were used. After multiple fractional intravenous (IV) injections in rats with cholangiocarcinoma, the tumours were irradiated through the skin by an 808-nm NIR diode laser at a power density of 2.3 W/cm2 for 15 min. For PDT, galactose – Luphthalocyanine, as a photosensitizer, was applied by intratumoural injection, in a dose of 2 mg/kg. Then, tumors were irradiated through the skin by 670 nm-diode laser source with power density 200 mW/cm2 applied for 1000 sec in three non-overlapping zones covering whole tumour surface to obtain a total irradiation dose of 200 J/cm2 for each lesion. The withdrawal of the animals from the experiment and sampling of tissues for morphological study were performed before and 72 hrs after PPT and PDT. The vascular microdensity in tumors was assessed on histological sections as vessel counts or vessel area per unit of assessed tumor area. It was shown that efficiency of PPT and PDT therapy was mostly due to the sufficient accumulation of photothermosensitizers in the tumor, therefore preliminary assessment of tumor vascularization degree was necessary before starting a therapy.

The influence of long-term peroral injection of gold nanoparticles with various sizes on the liver, spleen, and lymph nodes of adult laboratory rats and their progeny was studied. The morphometric analysis was conducted using a µVizo-103 medical digital microviser. The conglomerated gold nanoparticles were detected in tissues via the dark-field microscopy. Gold nanoparticles with sizes of 2 and 50 nm were found to cause the mostly pronounced morphological alterations in organs that testifies to their size-dependent impact. The liver in a group receiving 50-nm gold particles exhibit noticeable dystrophy of hepatocytes and activation of a monocyte macrophage system. The spleen and lymph nodes reveal the activation of proliferation and differentiation of immunocompetent cells and macrophages. Peroral injection of gold particles during gestation of females led to an increase in a number of hepatocytes with necrobiotic changes as well as to elevated amount of non-parenchymatic elements in the liver of the progeny, especially in groups containing the 2- and 50-nm gold nanoparticles.

The possibilities of using fluorescence-based analytical methods and their advantages for visualization and identification of the type of programmed cell death in human tumor cells exposed to flavonoids have been analyzed in experiments in vitro. HeLa cervical cancer cells and A498 kidney carcinoma cells were used as the objects of the study, and exposure to the flavonoid-containing extract of common hedgehyssop (Gratiola officinalis L.) was tested as the experimental treatment. The following fluorescence-based techniques were used: the “live and dead” test with double staining by propidium iodide and acridine orange and double staining with annexin and propidium iodide. Autophagy induction was confirmed by fluorescence-based tests implemented in a Muse cell analyzer with a Muse Autophagy LC3-Antibody Based Kit. The use of double staining with acridine orange and propidium iodide fluorescent dyes in the “live and dead” test and comparison with phase contrast microscopy enables the visualization of apoptotic body and autophagosome formation processes in the cells and can, therefore, be used as a method of screening assessment of the efficiency of various chemotherapy drugs.

The extract of G. officinalis was separated by five major fractions and the antitumor activity of hydrophobic fractions has been established. For the most active fractions spectral investigations of the main component were performed using UV-Vis electronic spectra, circular dichroism (CD), Fourier-transform infrared spectroscopy (FTIR) and nuclear magnetic resonance (NMR) spectroscopy. On the basis of spectral characteristics, it was demonstrated that the main components of the fractions 4 and 5 of G. officinalis providing its biological activity, highly likely, are of flavonoid nature. Based on the NMR data we propose the structure of the aglycon moiety of the main component from fraction 4 as 4,2’,5’-trihydroxychalcone. The fraction 5 is the most active, the antitumor effect of which is implemented through cytotoxic, cytostatic, apoptotic and autophagosomal activity in respect to human kidney cancer tumor cells.

The tumor vascularization in rats with transplanted cholangiocarcinoma was assessed by US power Doppler before plasmonic photothermal therapy (PPT). For PPT, the multiple intravenous injections of PEG-coated gold nanorods and irradiation by 808-nm NIR diode laser were used. We have shown that gold nanoparticles accumulation in tumor and efficiency of PPT are associated with the degree of vascularization of a tumor.

The heating degree of the inner layers of tumor tissue is an important parameter required to optimize plasmonic photothermal therapy (PPT). This study reports the optical properties of tissue layers of transplanted cholangiocarcinoma and covering tissues in rats without treatment (control group) and after PPT using gold nanorods (experimental group). PPT was carried out for 15 min, and the temperature on the skin surface reached 54.8 ± 1.6 °C. The following samples were cut out ex vivo and studied: skin, subcutaneous connective tissue, tumor capsule, top, center, and bottom part of the tumor. The samples’ absorption and reduced scattering coefficients were calculated using the inverse adding–doubling method at 350–2250 nm wavelength. Diffuse reflectance spectra of skin surface above tumors were measured in vivo in the control and experimental groups before and immediately after PPT in the wavelength range of 350–2150 nm. Our results indicate significant differences between the optical properties of the tissues before and after PPT. The differences are attributed to edema and hemorrhage in the surface layers, tissue dehydration of the deep tumor layers, and morphological changes during the heating.

Two models of diabetes, such as alloxan and streptozotocin, were used in the study to verify the influence of both on the optical properties and molecular permeability of skin ex vivo. Kinetics of collimated transmittance of control and diabetic skin samples immersed in aqueous 70%-glycerol solution was measured and then processed for estimation of diffusion coefficient of glycerol in skin. As a result, the slower glycerol diffusion rate was observed for both models of diabetes.

The study of blood microcirculation is one of the most important problems of the medicine. This is caused by the fact that many diseases, such as cardio-vascular diseases, atherosclerosis, diabetes, chronic venous insufficiency, oncology diseases, cause functional and morphological changes of microcirculation of blood flow. The results of experimental study of changes of blood microcirculation of pancreas in rats with diabetes measured by using Laser Speckle Contrast Imaging (LSCI) at application of optical clearing agents are presented. Laser speckle contrast techniques are based on the spatial and temporal statistics of the speckle pattern, calculating of contrast of time-averaged dynamic speckles in dependence on the exposure time at the registration of the speckle-modulated images. In research, 38 Wistar rats weighing 300-500 g were used. Alloxan induced animal model and streptozotocin model of diabetes was explored. The influence of solution of glycerol, Omnipaque®-300 was investigated. Application of 70%-aqueous glycerol solution demonstrates 50%-decrease of blood flow velocity in the group of aloxan diabetic animals, to 10th min blood flow velocity was completely restored. In a group of animals with streptozotocin diabetes, the blood flow rate decreased by 54%, by 10 minutes the speed did not reach the initial value. Blood flow in the control group almost stopped, to 10 min has not recovered. Application of Omnipaque®-300 demonstrates 65%-increase of blood flow in the group of alloxan diabetic animals and an increase of bloode flow in the first 2 minutes of applying the solution by 45%. In both cases to 10th min, blood flow velocity was completely restored. Blood flow in the control group did not show any noticeable changes.

In this report, the influence of C. psittaci (CP) antigens on the process of a development of a malignant solid tumor in the inbred Wistar white rats in experimental conditions has been successfully studied. Velocity of growing of a tumor has been estimated by s-LASCA technique. Measurement of statement of a tumor has been performed daily after each of three serial injections of the CP specific ornithosis antigen derived from two different CP strains, the Rostinovo-70 and the АМК. Single injection solutions were administered in a volume of 1 ml. We found, that both antigens could initiate the marked decrease of the growth of malignant tumors in the animal model used.

The aim of the study is investigation of morphological features of the testicles of rat newborns with different models of intrauterine hypoxia. Fifteen white outbred female rats aged from 4 to 10 months with a weight of 200 ± 30 g were used. Laboratory animals were divided into 3 experimental groups, 5 rats in each. The first group was subjected to a hypoxic hypoxia during pregnancy (21 days). The hypoxia was simulated in accordance with the method of N.N. Karkishchenko (2010). The second group was subjected to a hemic hypoxia during the second and third weeks of pregnancy, in accordance with the methodology of L.M. Sosedova (2012). The third (control) group was not exposed to any treatment during the pregnancy. After delivery, the newborn rat pups were subjected to the measurements of oxygenation degree using the methods of reflectance spectroscopy, then the animals were withdrawn from the experiment; and a morphological study of the testicle tissues was performed. Histological examination of the tissues of the testicles of newborn rats showed signs of impaired blood circulation, dystrophic and necrobiotic changes in the parenchyma.

Application of many optical methods, such as photoacoustics, optical coherent tomography, and terahertz spectroscopy, for tissue imaging and disease diagnostics is limited due to small probing depth caused by strong scattering and absorption in tissues [1, 2]. It is possible to increase the efficiency of optical techniques by using tissue optical clearing (TOC), which allows one to control tissue optical properties by exposing tissue to biocompatible liquids - optical clearing agents (OCAs). When exposed to an OCA with hyperosmotic properties, such as glycerol or highly concentrated glucose solution, it is possible to achieve a considerable decrease of tissue scattering within the entire spectral range and absorption on the water bands due to refractive index matching phenomenon and dehydration of the tissue, and therefore to get a significant increase of the optical and THz wave transmittance [1-3]. In vitro studies were performed on laboratory rats of the Vistar strain (sexually mature females weighing 300–400 g were used). The animals were inoculated with the tumor cells by subcutaneous injection into the area of the scapulae. Studies were conducted in 28 days after injection. The weight and thickness of the samples were measured before and after optical clearing by using a micrometer with an accuracy of ±0.001 mm and an analytical balance (Scientech, SA210, USA) with an accuracy of ±1 mg. The samples of the area 1.5*1.5 cm2 were placed in a closed sealed container, to which was added 2 ml of OCA. 70% glycerol solution, 40% glucose solution and 50% fructose in a 30% alcohol solution were used with the addition of saline as an OCA. The transmission spectrum of glycerol in the frequency range of 0.05–1.0 THz was measured by using the terahertz (THz) time-domain spectrometer. Measurements of the refractive index of OCA were carried out before the start of the experiment and every 5 minutes after placing the skin sample in the solution with OCA for 15 minutes, then every 10 minutes for another 45 minutes. It was found that the effectiveness of optical clearing of the skin over the tumor is higher compared to healthy skin. A good correlation was shown between the kinetics of the sample mass and THz absorption and refraction. An assessment of skin dehydration also showed that the concentration of water extracted from the skin above the tumor is greater compared to healthy. It is especially important that enhancing the image contrast due to dehydration and TOC in the pathology area is extremely significant in diagnosing precancerous conditions and treating early stages of tumor, due to the possibility of selective increase of the scattering contrast of cell nuclei relative to surrounding cytoplasm and interstitial fluid and thus following their size and internal rearrangements [5].

The new generations of photoactive compounds, which are derivatives of already well-established groups of photosensitizers, together with the modern light sources in the spectral region of their activation and in the PDT window range, are emerging as a reliable phototherapy approach for tumour treatment with higher efficiency. The substitution of a highly hydrophobic phthalocyanine with galactose units could improve the solubility and to decrease significant aggregation in polar solvents. The control group of rats with transplanted cholangiocarcinoma without any treatment was used for comparison of PDT effectiveness. In the second group the white outbred male rats with cholangiocarcinoma lesions were treated using Zn-phthalocyanine, as a typical representative of phthalocyanine photosensitizers with known photodynamic properties and the third group of animals was treated with galactose – Lu-phthalocyanine, as a novel derivative from the same family with two specific differences – presence of galactose and replacing of Zn(II) with Lu(III) ion. To compare the PDT effectiveness of both compounds the same drug doses were applied - 2 mg/kg, applied by intratumoural injection. Diode laser source at 670 nm, on 50 mW output power with power density applied 200 mW/cm2 was applied for 1000 sec to obtain total irradiation dose of 200 J/cm2 for each lesion. 3 days after treatment, the animals were withdrawn from the experiment. Tissue from the central zone and periphery of the tumor was taken for morphological examination and fixed in 10% formalin. Serial paraffin sections were stained with hematoxilin-eosin and by immunohistochemical staining with antibodies to proliferation marker Ki-67 and apoptosis marker BAX. After photodynamic therapy, pronounced necrobiotic changes in combination with an inflammatory reaction were developed at the central zone of tumors. The dystrophic changes, reduction of proliferation and development of apoptosis in tumor cells were observed on tumor periphery as well.

Modern optical biomedical technologies require a comprehensive knowledge of the optical properties of both healthy and tumor tissues in a wide range of wavelengths. Morphological and biochemical changes associated with the development of cancer in many cases depend on the specific type and location of the cancer. This chapter discusses the optical and physiological properties of various types of normal, benign, and malignant tissues of lung, breast, colorectal, prostate, cervical, bladder, stomach, esophageal, liver, kidney, skin, oral, thyroid, and brain obtained in vitro, ex vivo, and in vivo using diffuse reflectance spectroscopy, fluorescence and Raman spectroscopy. Biochemical diagnostic models were analyzed as a tool that helps determine tissue biochemical composition and functional state of cellular metabolism. The data obtained can be used for the development of novel methods of cancer diagnostics and treatment.

Background The analysis of recent studies on plasmonic photothermal therapy (PPT) after intravenous administration of gold nanorods (GNRs) has demonstrated that the effectiveness of nanoparticle-assisted laser hyperthermia depends on a correct dosage strategy of nanoparticle administration. Accumulation of GNRs in tumor tissue dramatically increases the local heating of the tumor without damage to healthy tissues. However, the optimal doses of GNR intravenous injections (IVIs) for effective accumulation in tumors, and optimal protocols of PPT are not designed yet. The current study aims to improve the efficacy of PPT in tumor-bearing rats using multiple fractional intravenous administration of GNRs. Materials and methods For PPT experiments, the GNRs with aspect ratio of 4.1 were functionalized with thiolated polyethylene glycol (PEG) and their suspensions were used for multiple fractional intravenous administration in outbred albino male rats with experimental model of rat liver cancer (cholangiocarcinoma line PC-1). Doppler ultrasonography was performed to characterize the vascularity of transplanted rat tumors before any treatment. After a final injection of GNRs, tumor was irradiated during 15 minutes by 808-nm NIR diode laser at a power density 2.3 W/cm2 . The animals were withdrawn from the experiment and sampling of tissues for morphological study and gold accumulation was performed 24 hours and 3 weeks after PPT. Results The multiple IVIs of gold nanorods and further PPT of transplanted cholangiocarcinoma provided significant damage to tumor tissue resulting in pronounced necrotic mass and retardation of the tumor growth. More importantly, the proposed PPT protocol had low toxicity as evidenced by histological examination of internal organs. The efficiency of PPT depends on the presence of newly formed vasculature as revealed by the Doppler ultrasound investigation. Conclusion The repeatable IVIs promote greater of GNR accumulation within the tumor thus resulting in higher PPT efficacy. Accompanying ultrasonography can be useful for prognosis and monitoring of treatment. Lasers Surg. Med. 50:1025-1033, 2018. © 2018 Wiley Periodicals, Inc.

Regretfully, because of her sudden demise, Assoc. Prof. Ekaterina Borisova is no longer amongst us. COVID-19 pulled away a brilliant scientist during the peak of her scientific career (see Fig. 1). All authors would like to express deepest condolences and sincere support to her family, friends, relatives and colleagues! We, therefore, rightfully commemorate her dedicated and devoted contribution to biophotonics, her readiness to always support, help, motivate and inspire all her colleagues and collaborators., We would like to acknowledge the support from the National Science Fund of the Bulgarian Ministry of Education and Science (NSFB-MES) under all current projects involving Assoc. Prof. Ekaterina Borisova, which are grant numbers: #DN 18-8; #КP06-N29/11/18.12.18; #KP06-N28/11/14.12.2018; #KP06-Russia-9/2020; #KP-06-N23/8; #KP-06/N38/13; #KP06-India-8/07.08.2019; #KP-06-KOST-6. Also, projects under NRRI #DO1-401/18.12.2020 and #D01-392/18.12.2020.

The chemical composition of a new bioactive extract from immortelle sandy (Helichrysum arenarium (L.) Moench.) was investigated. Naringin, its soluble aggregate, prunin, quercetin, apigenin, naringenin, apigenin 5-O-glucoside, and isosalipurposide were found among the flavonoids. The tested immortelle sandy extract was shown to contain known dimers, trimers, or more complex aggregates. The compounds had similar spectral but substantially different chromatographic characteristics, which can be used for their identification based on the corresponding data on flavonoids glycosylated at different positions and/or carbohydrates of various complexities, which are also characterized by similar absorption spectra and different retention times. It was found by molecular absorption spectroscopy that the immortelle extract contained 73.48 mg of flavonoids relative to rutin or 17.94 mg relative to quercetin per 1 g of dry extract weight, which corresponded to 20.99 and 5.13%, respectively. The immortelle extract obtained by the method proposed displayed antitumor activity against the transplanted sarcoma 45 and had a beneficial effect on animals in general.

Optical methods with highly sensitive to water content are important for the development of new diagnostic and therapeutic methods in medicine. The paper presents a quantitative assessment of the dehydration of skin when using optical clearing agents (glycerol and glucose) by the method of multi-wavelength refractometry for the visible and NIR spectral regions. The resulting decrease in the refractive index of the optical clearing solution made it possible to estimate the volume of the fluid extracted from the tissue. The possibility of using the method when conducting in vivo measurements is shown. An assessment was made of the degree of dehydration of rat skin in areas with a subcutaneous tumor neoplasm, which was three times less than for control (healthy) skin areas.

The main objective of this work is to quantify the impact of photodynamic/photothermal treatment by using visible LED and NIR laser irradiation through the skin of subcutaneous fat in vivo followed up by tissue sampling and histology. The optical method may provide reduction of regional or site-specific accumulations of abdominal or subcutaneous adipose tissue precisely and least-invasively by inducing cell apoptosis and controlled necrosis of fat tissue. As photodynamic/photothermal adipose tissue sensitizers Brilliant Green (BG) or Indocyanine Green (ICG) dyes were injected subcutaneously in rats. The CW LED device (625 nm) or CW diode laser (808 nm) were used as light sources, respectively. Biopsies of skin together with subcutaneous tissues were taken for histology. The combined action BG-staining and LED-irradiation (BG + LED) or ICG-staining and NIR-laser irradiation (ICG + NIR) causes pronounced signs of damage of adipose tissue characterized by a strong stretching, thinning, folding and undulating of cell membranes and appearance of necrotic areas. As a posttreatment after 14 days only connective tissue was observed at the site of necrotic areas. The data obtained are important for safe light treatment of site-specific fat accumulations, including cellulite. This work provides a basis for the development of fat lipolysis technologies and to move them to clinical applications. Schematics of animal experiment.

To establish the mechanisms of pathomorphism of transplantable kidney cancer in rats that used flavonoid-containing hedge hyssop (Gratiola officinalis L.) extract in an in vivo experiments.The experiment was carried out on 30 male Wistar rats with transplantable kidney cancer PA. At 72 hours after tumor inoculation, the rats in the experimental groups received hedge hyssop extract at an oral or intramuscular dose of 110 mg/kg/day for 12 days. A comparison group consisted of the animals with a tumor, but without exposure. The investigators used the immunohistochemical markers of apoptosis (p53, bax, bcl-2, Fas-receptor, Fas-ligand), autophagy (LC3B), proliferation (Ki-67), and angiogenesis (VEGF). During statistical data processing, the Shapiro-Wilk test was used to test the normality of the indicator distribution in the groups. Cramer-Welch's t-test was also employed to compare the groups.Histological examination of tumor tissue under the action of hedge hyssop extract showed the emergence of extensive areas of damage (necrosis and apoptotic bodies). With both routes of hedge hyssop extract administration, there was a sharp decrease in the expression of the proliferation markers Ki-67 and the angiogenesis marker VEGF and a high expression of the apoptosis markers p53, bax, CD95 (Fas/APO-1), and FAS-ligand in tumor cells and its absence in the comparison group. All the described changes were more pronounced with intramuscular administration. The expression of the autophagy marker LC3B increased with the oral administration of hedge hyssop extract and decreased with its intramuscular administration.A pronounced pathomorphism of kidney cancer develops due to consumption of hedge hyssop extract. This suppresses the proliferation, angiogenesis, and activation of apoptotic signaling and mitochondrial pathways and blocks protective autophagy. The autophagy marker LC3B can be used as an additional criterion for evaluating the therapeutic pathomorphism of tumors.Цель исследования - в экспериментах in vivo установить механизмы патоморфоза перевитого рака почки под влиянием флавоноидсодержащего экстракта аврана. Материал и методы. Эксперимент проводили на 30 самцах крыс Wistar c перевитым раком почки РА. В опытных группах крысы через 72 ч после перевивки опухоли на протяжении 12 сут получали экстракт аврана перорально или внутримышечно в дозе 110 мг/кг/сут. Группу сравнения составили животные с опухолью, но без воздействия. Использовали иммуногистохимические маркеры апоптоза (p53, bax, bcl-2, Fas-receptor, Fas-ligand), аутофагии LС3b, пролиферации Ki-67 и ангиогенеза VEGF. При статистической обработке данных нормальность распределения показателей в группах проверяли при помощи критерия Шапиро-Уилка. Для сравнения групп использовали критерий Крамера-Уэлча (Т). Результаты. При гистологическом исследовании в ткани опухоли под действием экстракта аврана наблюдали появление обширных зон повреждения (некроз и апоптозные тельца). При обоих путях введения экстракта аврана отмечали резкое снижение экспрессии маркеров пролиферации (Ki-67), ангиогенеза (VEGF) и высокую экспрессию маркеров апоптоза (p53, bax, CD95 (Fas/APO-1), FAS-ligand) в опухолевых клетках и отсутствие ее в группе сравнения. Все описанные изменения были более выражены при внутримышечном введении. Экспрессия маркера аутофагии LC3B увеличивалась при пероральном и уменьшалась при внутримышечном введении экстракта аврана. Заключение. Под влиянием экстракта аврана развивается выраженный патоморфоз рака почки. Происходят подавление пролиферации, ангиогенеза и активация сигнального и митохондриального пути апоптоза, а также блокирование протекторной аутофагии. Маркер аутофагии LC3b может использоваться в качестве дополнительного критерия оценки лечебного патоморфоза опухолей.

The study of blood microcirculation is one of the most important problems of the medicine. This is caused by the fact that many diseases, such as cardio-vascular diseases, atherosclerosis, diabetes, chronic venous insufficiency, oncology diseases, cause functional and morphological changes of microcirculation of blood flow. The results of experimental study of changes of blood microcirculation of pancreas in rats with diabetes measured by using Laser Speckle Contrast Imaging (LSCI) at application of optical clearing agents are presented. Laser speckle contrast techniques are based on the spatial and temporal statistics of the speckle pattern, calculating of contrast of time-averaged dynamic speckles in dependence on the exposure time at the registration of the speckle-modulated images. In research, 28 Wistar rats weighing 300-500 g were used. Alloxan induced animal model of diabetes was explored. The influence of solution of glycerol, PEG-300 was investigated. Application of 70%-aqueous glycerol solution demonstrates 50%-decrease of blood flow velocity in the group of diabetic animals, to 10th min blood flow velocity was completely restored. Blood flow in the control group almost stopped, to 10 min has not recovered. Application of solution of PEG-300 demonstrates 25%- decrease of blood flow in the group of diabetic animals. Blood flow in the control group show 65%-decrease of blood flow. The results obtained at the study of blood microcirculation disorders of pancreas in diabetes show that diseases development in animals causes changes in the microcirculatory system and application of optical clearing agents demonstrates changes in vascular permeability in conditions of development of pathologies.

The study offers a possibility to use such biocompatible chemical as glycerol as a biomarker for early diagnostics of diabetes mellitus complications, in particular, for assessing the degree of myocardial lesion evidence by the stage of skin glycation during diabetes development and treatment. The study was aimed to find the difference in glycerol permeability through rat soft tissues of control and diabetic groups in order to show the possibility of glycerol usage as a biomarker of diabetes impact on different organs. The investigation presents transition the experimental studies from ex vivo to in vivo conditions.

The aim of work was to study the characteristics of uteroplacental blood flow in pregnant rats, tissue oxygenation and the morphological features of the main internal organs (heart, liver, kidneys) of newborns during chronic intrauterine hypoxia. The rats (8 animals) were divided into 2 experimental groups. The first (hypoxia) group was subjected to hypoxia throughout pregnancy (21 days). The second (control) group was not exposed to any effect throughout the pregnancy. On the 20th day of gestation in both groups, a Doppler ultrasound study of uterine-fetal blood flow and measurement of the placenta was performed. Newborn rat pups of the first day of life were subjected to oxygenation degree measurement. After delivery, the number of rats in the litter was counted and their body weight was measured. After the measurements the laboratory animals were withdrawn from the experiments and morphological study of their internal organs was performed. The studies have shown a clear relationship between the characteristics of uterine blood flow and the presence or absence of hypoxia. In chronic hypoxia, there were low rates of arterial blood flow, an increase in the peripheral resistance of uterine vessels. All this indicates the defective formation of the placenta and impaired blood flow with the development of placental insufficiency. It was also found that hypoxia in the antenatal period adversely affects the number and somatometric indicators of newborn rats in the offspring. Histological examination of the tissues of the heart, liver and kidney of newborn rat pups showed signs of impaired blood circulation, dystrophic and necrobiotic changes in the parenchyma.

Terahertz time-domain spectroscopy (THz-TDS) in the 0.05–1.0 THz frequency range was used to study the blood serum of rats in the dynamics of experimental liver cancer. It was shown that the THz transmission spectra of samples from healthy animals and 14 and 28 days after tumor cells implantation are similar in shape, but differ in amplitude. There is a change in the biochemical composition of the blood, including a decrease in the protein content, by the 28th day of the experiment. The observed changes in the THz response are explained by a decrease in the number of protein molecules and bound water and correlate with changes in the refractive index in the visible and near-IR ranges from 480 to 1550 nm, measured using a multiwave refractometer.