Characteristics of the Antitumor Effect of Doxorubicin and Pegylated Hyaluronidase on Models of Rat Brain Tumors.

Hyaluronidase increases tissue permeability and diffusion of the extracellular fluid by cleaving hyaluronan, the primary component of the extracellular matrix. Hyaluronidase pegylation (Hyal-PEG) decreases its clearance and enhances biodistribution. The pro- and anticancer activity of Hyal-PEG and a combination of Hyal-PEG with doxorubicin were studied in vitro (morphological analysis of rat glioblastoma 101.8 spheroids) and in vivo (by the survival time of rats after intracerebral transplantation of the tumor and morphological analysis). In the presence of doxorubicin and Hyal-PEG in the culture medium in vitro, spheroids lost their ability to adhere to the substrate and disintegrate into individual cells. Intracerebral transplantation of the tumor tissue with Hyal-PEG did not accelerate glioblastoma growth. The mean survival time for animals receiving transplantation of the tumor alone and in combination with Hyal-PEG was 13 and 20 days, respectively. In one rat with transplanted tumor and Hyal-PEG, this parameter increased by 53%. The survival time of rats receiving systemic therapy with doxorubicin and Hyal-PEG significantly increased (p=0.003). Antitumor effect of therapeutic doses of doxorubicin combined with Hyal-PEG was demonstrated on the model of rat glioblastoma 101.8 in vitro. Hyal-PEG inhibited adhesion of tumor cells, but did not cause their death. Transplantation of Hyal-PEG-treated tumor did not reduce animal survival time. Systemic administration of therapeutic doses of doxorubicin with Hyal-PEG increased survival time of rats with glioblastoma 101.8.

Comparative Morphological and Molecular Genetic Characteristics of Cell and Tissue Strains of Experimental Rat Glioma 10-17-2 (Astrid-17).

In order to obtain models of gliomas of varying degrees of malignancy, we performed morphological and molecular genetic study of a tissue strain of glioma 10-17-2 (Astrid-17) obtained by intracranial passaging of tumor fragments of chemically induced rat brain tumor, and a cell strain isolated from it. More or less pronounced changes in the expression levels of Mki67, Trp53, Vegfa, and Gfap genes in the tissue and cell strain of glioma 10-17-2 (Astrid-17) compared with intact brain tissue were shown. The tissue model of glioma 10-17-2 (Astrid-17) according to the studied characteristics shows features of grade 3-4 astrocytoma and the cellular model - grade 2-3 astrocytoma.

The Effect of Therapy Regimen on Antitumor Efficacy of the Nanosomal Doxorubicin against Rat Glioblastoma 101.8.

One of the key problems of glioblastoma treatment is the low effectiveness of chemotherapeutic drugs. Incorporation of doxorubicin into PLGA nanoparticles allows increasing the antitumor effect of the cytostatics against experimental rat glioblastoma 101.8. Animal survival, tumor volume, and oncogene expression in tumor cells were compared after early (days 2, 5, and 8 after tumor implantation) and late (days 8, 11, and 14) start of the therapy. At late start, a significant increase in the expression of oncogenes Gdnf, Pdgfra, and Melk and genes determining the development of multidrug resistance Abcb1b and Mgmt was revealed. At early start of therapy, only the expression of oncogenes Gdnf, Pdgfra, and Melk was enhanced. Early start of treatment prolonged the survival time and increased tumor growth inhibition by 141.4 and 95.7%, respectively, in comparison with the untreated group; these differences were not observed in the group with late start of therapy. The results indicate that the time of initiation of therapy is a critical parameter affecting the antitumor efficacy of DOX-PLGA.

Quantification of attenuation and speckle features from endoscopic OCT images for the diagnosis of human brain glioma.

Application of optical coherence tomography (OCT) in neurosurgery mostly includes the discrimination between intact and malignant tissues aimed at the detection of brain tumor margins. For particular tissue types, the existing approaches demonstrate low performance, which stimulates the further research for their improvement. The analysis of speckle patterns of brain OCT images is proposed to be taken into account for the discrimination between human brain glioma tissue and intact cortex and white matter. The speckle properties provide additional information of tissue structure, which could help to increase the efficiency of tissue differentiation. The wavelet analysis of OCT speckle patterns was applied to extract the power of local brightness fluctuations in speckle and its standard deviation. The speckle properties are analysed together with attenuation ones using a set of ex vivo brain tissue samples, including glioma of different grades. Various combinations of these features are considered to perform linear discriminant analysis for tissue differentiation. The results reveal that it is reasonable to include the local brightness fluctuations at first two wavelet decomposition levels in the analysis of OCT brain images aimed at neurosurgical diagnosis.

[Covalently conjugated DNA aptamer with doxorubicin as in vitro model for effective targeted drug delivery to human glioblastoma tumor cells]. / Kovalentno kon"yugirovannyi DNK-aptamer s doksorubitsinom kak in vitro model' effektivnogo adresnogo vozdeistviya na opukholevye kletki glioblastomy cheloveka.

Targeted delivery of chemotherapeutic agents with aptamers is a very effective method increasing therapeutic index compared to non-targeted drugs. OBJECTIVE: To study the effectiveness of in vitro therapeutic effect of covalently conjugated GR20 DNA aptamer with doxorubicin on glioblastoma cells compared to reference culture of human fibroblasts. MATERIAL AND METHODS: A Sus/fP2 cell culture was obtained from glioblastoma tissue sample to analyze the effectiveness of conjugate. A linear culture of human dermal fibroblasts (mesenchymal stem cells) DF1 was used as a control. To assess antiproliferative activity of covalently conjugated GR20 aptamer with doxorubicin, we used the MTS test. The Cell Index was measured using the xCelligence S16 cell analyzer assessing viability of cell cultures by recording changes in real time. RESULTS: Human glioblastoma Sus/fP2 cells reduce own proliferative potential by 80% when exposed to doxorubicin (0.5 µM, 72 hours, MTS test), by 9% when exposed to GR20 aptamer (10 µM, 72 hours, MTS test) and by 26% when exposed to covalently conjugated DOX-GR20 (0.5 µM, 72 hours, MTS test). A long-term study of proliferative potential of Sus/fP2 cells on the xCelligence S16 analyzer revealed a significant decrease in the number of cells under the effect of doxorubicin and covalently conjugated DOX-GR20. Effectiveness of covalently conjugated DOX-GR20 is halved. GR20 aptamer at a concentration of 10 µM and its conjugate with doxorubicin DOX-GR20 at a concentration of 1 µM have no negative effect on cells of the control culture of DF1 fibroblasts, while doxorubicin is toxic for these cells. MTS test and xCelligence S16 cell analyzer found no decrease in metabolic activity of DF1 cells and their ability to proliferate. CONCLUSION: We established obvious antiproliferative effect of covalent conjugate DOX-GR20 on continuous human glioblastoma cell culture Sus/fP2 without toxic effect on the reference culture (dermal fibroblasts DF1).

Quantitative polarization-sensitive super-resolution solid immersion microscopy reveals biological tissues' birefringence in the terahertz range.

Terahertz (THz) technology offers a variety of applications in label-free medical diagnosis and therapy, majority of which rely on the effective medium theory that assumes biological tissues to be optically isotropic and homogeneous at the scale posed by the THz wavelengths. Meanwhile, most recent research discovered mesoscale ([Formula: see text]) heterogeneities of tissues; [Formula: see text] is a wavelength. This posed a problem of studying the related scattering and polarization effects of THz-wave-tissue interactions, while there is still a lack of appropriate tools and instruments for such studies. To address this challenge, in this paper, quantitative polarization-sensitive reflection-mode THz solid immersion (SI) microscope is developed, that comprises a silicon hemisphere-based SI lens, metal-wire-grid polarizer and analyzer, a continuous-wave 0.6 THz ([Formula: see text] µm) backward-wave oscillator (BWO), and a Golay detector. It makes possible the study of local polarization-dependent THz response of mesoscale tissue elements with the resolution as high as [Formula: see text]. It is applied to retrieve the refractive index distributions over the freshly-excised rat brain for the two orthogonal linear polarizations of the THz beam, aimed at uncovering the THz birefringence (structural optical anisotropy) of tissues. The most pronounced birefringence is observed for the Corpus callosum, formed by well-oriented and densely-packed axons bridging the cerebral hemispheres. The observed results are verified by the THz pulsed spectroscopy of the porcine brain, which confirms higher refractive index of the Corpus callosum when the THz beam is polarized along axons. Our findings highlight a potential of the quantitative polarization THz microscopy in biophotonics and medical imaging.

Gene Expression Profile of 3D Spheroids in Comparison with 2D Cell Cultures and Tissue Strains of Diffuse High-Grade Gliomas.

The use of relevant, accessible, and easily reproducible preclinical models of diffuse gliomas is a prerequisite for the development of successful therapeutic approaches to their treatment. Here we studied the gene expression profile of 3D spheroids in a comparison with 2D cell cultures and tissue strains of diffuse high-grade gliomas. Using real time PCR, we evaluated the expression of Gfap, Cd44, Pten, S100b, Vegfa, Hif1a, Sox2, Melk, Gdnf, and Mgmt genes playing an important role in the progression of gliomas and regulating tumor cell proliferation, adhesion, invasion, plasticity, apoptosis, DNA repair, and recruitment of tumor-associated cells. Gene expression analysis showed that 3D spheroids are more similar to tumor tissue strains by the expression levels of Gfap, Cd44, and Pten, while the expression levels of Hif1a and Sox2 in 3D spheroids did not differ from those of 2D cell cultures, the expression levels S100b and Vegfa in 3D spheroids was higher than in other models, and the expression levels of Melk, Gdnf, and Mgmt genes changed diversely. Thus, 3D spheroid model more closely mimics the tumor tissue than 2D cell culture, but still is not the most relevant, probably due to too small size of spheroids, which does not allow reproducing hypoxia and apoptotic and necrotic processes in the tumor tissue.

Morphological and molecular-biological features of glioblastoma progression in tolerant and susceptible to hypoxia Wistar rats.

Hypoxia is a major pathogenetic factor in many cancers. Individual resistance to suboptimal oxygen availability is subject to broad variation and its possible role in tumorigenesis remains underexplored. This study aimed at specific characterization of glioblastoma progression in male tolerant and susceptible to hypoxia Wistar rats. Hypoxia resistance was assessed by gasping time measurement in an 11,500 m altitude-equivalent hypobaric decompression chamber. Based on the outcome, the animals were assigned to three groups termed 'tolerant to hypoxia' (n = 13), 'normal', and 'susceptible to hypoxia' (n = 24). The 'normal' group was excluded from subsequent experiments. One month later, the animals underwent inoculation with rat glioblastoma 101.8 followed by monitoring of survival, body weight dynamics and neurological symptoms. The animals were sacrificed on post-inoculation days 11 (subgroup 1) and 15 (subgroup 2). Relative vessels number, necrosis areas and Ki-67 index were assessed microscopically; tumor volumes were determined by 3D reconstruction from histological images; serum levels of HIF-1α, IL-1ß, and TNFα were determined by ELISA. None of the tolerant to hypoxia animals died of the disease during observation period, cf. 85% survival on day 11 and 55% survival on day 15 in the susceptible group. On day 11, proliferative activity of the tumors in the tolerant animals was higher compared with the susceptible group. On day 15, proliferative activity, necrosis area and volume of the tumors in the tolerant to hypoxia animals were higher compared with the susceptible group. ELISA revealed no dynamics in TNFα levels, elevated levels of IL-1ß in the susceptible animals on day 15 in comparison with day 11 and tolerant ones. Moreover, there were elevated levels of HIF-1α in the tolerant animals on day 15 in comparison with day 11. Thus, the proliferative activity of glioblastoma cells and the content of HIF-1α were higher in tolerant to hypoxia rats, but the mortality associated with the tumor process and IL-1ß level in them were lower than in susceptible animals. Specific features of glioblastoma 101.8 progression in tolerant and susceptible to hypoxia rats, including survival, tumor growth rates and IL-1ß level, can become the basis of new personalized approaches for cancer diseases treatment in accordance to individual hypoxia resistance.

Changes in Oncogene Expression in Experimental Glioblastoma 101.8 Rats during Therapy with PLGA Nanoparticles Loaded with Doxorubicin.

We compared the expression of the main glioblastoma oncogenes during therapy with doxorubicin (Dox) and Dox in nanoparticles based on a copolymer of lactic and glycolic acids (Dox-PLGA) at a delayed start of treatment. Late initiation of Dox-PLGA therapy of glioblastoma showed an increase in the expression of multiple drug resistance genes, such as Abcb1b and Mgmt, and a decrease in Sox2 expression. Increased expression of other oncogenes (Melk, Wnt3, Gdnf, and Pdgfra) were observed during both Dox and Dox-PLGA therapy. These changes indicate increased tumor aggressiveness and its resistance to cytostatics at the late start of therapy.

[Nitric oxide donor nitrosorbide potentiates the antitumor effect of doxorubicin against experimental glioblastoma]. / Donor oksida azota izosorbida dinitrat povyshaet protivoopukholevyi effekt doksorubitsina pri khimioterapii eksperimental'noi glioblastomy.

BACKGROUND: Doxorubicin is a well-known antitumor drug that is not employed for chemotherapy of brain tumors. Indeed, doxorubicin does not penetrate across the blood-brain barrier in therapeutic concentrations. OBJECTIVE: To study the antitumor effect of doxorubicin combined with nitrosorbide on intracranial experimental glioblastoma 101/8 in rats. MATERIAL AND METHODS: Male Wistar rats (n=86) with intracranial implanted glioblastoma 101/8 received doxorubicin (i.v. 1.5 mg/kg thrice) alone or in combination with nitrosorbide (i.v or orally, 0.5 mg/kg thrice) in 2, 5 and 8 days after implantation. Efficacy was assessed considering survival and brain tumor volume in 14 days after tumor implantation. RESULTS: Combination of doxorubicin and nitrosorbide significantly increased survival (57% and 155%, respectively) and slowed down tumor growth (16±12 and 8±6 mm3, respectively) compared to doxorubicin alone. Effectiveness of nitrosorbide alone was trivial. CONCLUSION: Nitric oxide donor nitrosorbide considerably potentiated the antitumor effect of doxorubicin against intracranial 101/8 glioblastoma in rats.

Terahertz dielectric spectroscopy and solid immersion microscopy of ex vivo glioma model 101.8: brain tissue heterogeneity.

Terahertz (THz) technology holds strong potential for the intraoperative label-free diagnosis of brain gliomas, aimed at ensuring their gross-total resection. Nevertheless, it is still far from clinical applications due to the limited knowledge about the THz-wave-brain tissue interactions. In this work, rat glioma model 101.8 was studied ex vivo using both the THz pulsed spectroscopy and the 0.15λ-resolution THz solid immersion microscopy (λ is a free-space wavelength). The considered homograft model mimics glioblastoma, possesses heterogeneous character, unclear margins, and microvascularity. Using the THz spectroscopy, effective THz optical properties of brain tissues were studied, as averaged within the diffraction-limited beam spot. Thus measured THz optical properties revealed a persistent difference between intact tissues and a tumor, along with fluctuations of the tissue response over the rat brain. The observed THz microscopic images showed heterogeneous character of brain tissues at the scale posed by the THz wavelengths, which is due to the distinct response of white and gray matters, the presence of different neurovascular structures, as well as due to the necrotic debris and hemorrhage in a tumor. Such heterogeneities might significantly complicate delineation of tumor margins during the intraoperative THz neurodiagnosis. The presented results for the first time pose the problem of studying the inhomogeneity of brain tissues that causes scattering of THz waves, as well as the urgent need to use the radiation transfer theory for describing the THz-wave - tissue interactions.

Capability of physically reasonable OCT-based differentiation between intact brain tissues, human brain gliomas of different WHO grades, and glioma model 101.8 from rats.

Optical coherence tomography (OCT) of the ex vivo rat and human brain tissue samples is performed. The set of samples comprises intact white and gray matter, as well as human brain gliomas of the World Health Organization (WHO) Grades I-IV and glioma model 101.8 from rats. Analysis of OCT signals is aimed at comparing the physically reasonable properties of tissues, and determining the attenuation coefficient, parameter related to effective refractive index, and their standard deviations. Data analysis is based on the linear discriminant analysis and estimation of their dispersion in a four-dimensional principal component space. The results demonstrate the distinct contrast between intact tissues and low-grade gliomas and moderate contrast between intact tissues and high-grade gliomas. Particularly, the mean values of attenuation coefficient are 7.56±0.91, 3.96±0.98, and 5.71±1.49 mm-1 for human white matter, glioma Grade I, and glioblastoma, respectively. The significant variability of optical properties of high Grades and essential differences between rat and human brain tissues are observed. The dispersion of properties enlarges with increase of the glioma WHO Grade, which can be attributed to the growing heterogeneity of pathological brain tissues. The results of this study reveal the advantages and drawbacks of OCT for the intraoperative diagnosis of brain gliomas and compare its abilities separately for different grades of malignancy. The perspective of OCT to differentiate low-grade gliomas is highlighted by the low performance of the existing intraoperational methods and instruments.

[Comparative study of the effects of alpha-25-dihydroxycholecalciferol and 24, 25-dihydroxycholecalciferol on calcium-phosphorus metabolism and on bone tissue in experimental kidney insufficiency in rats]. / Sravnitel'noe izuchenie vliianiia lal'fa, 25-dioksikholekal'tsiferola i 24, 25-dioksikholekal'tsiferola na fosforno-kal'tsievyi obmen i kostnuiu tkan' pri éksperimental'noi pochechnoi nedostatochnosti u krys.

Experimental chronic kidney insufficiency (CKI; within 2-6 months) in rats, kept on a diet containing 0.6% Ca2+ and 0.6% P was accompanied by distinct azotemia, hyperphosphatemia, by a decrease in specific weight, in content of Ca2+, P and hydroxyproline in diaphyses as well as by a decrease in epiphyseal Ca2+. Daily administration of 0.025 micrograms of 1 alpha, 25-dihydroxy-cholecalciferol (1,25 (OH)2D3) into the animals did not normalize any of the patterns studied. At the same time, 1,25 (OH)2D3 increased the rate of hypercalciemia and demineralization of epiphyses, causing a slight hypercalciemia and increasing distinctly calcinosis of aorta as well as of the remaining part of the kidney. After daily administration of 24, 25-dihydroxycholecalciferol (24, 25 (OH)2D3) at a dose of 0.25 micrograms most of the patterns studied were normalized; specific weight, content of Ca2+ and P were increased in diaphyses simultaneously with a decrease in blood phosphorus concentration and in the level of azotemia. 24, 25 (OH)2D3 increased also the collagen content in diaphyses and epiphyses. The higher dose of 24, 25 (OH)2D3 (1.25 micrograms) did not exhibit higher effectivity. No one of the 24, 25 (OH)2D3 doses used did cause hypercalciemia and calcinosis. Combination of 0.025 micrograms 1,25 (OH)2D3 with 1.25 micrograms of 24, 25 (OH)2D3 decreased slightly the hypercalciemic, hyperphosphatemic and calcinosis inducing effects of 1,25 (OH)2D3 preventing completely the osteoporotic alterations in diaphyses but increasing the epiphysis demineralization; these results indicate that the doses of these metabolites must be decreased if their combination is required. The data obtained suggest that 24, 25 (OH)2D3 is a more effective and safe drug in correction of Ca2+-P metabolism impairments as well as of bone destruction under kidney insufficiency conditions as compared with 1,25 (OH)2D3.