Anna Maslennikova

Effects of gamma irradiation on collagen damage and remodeling

Abstract Purpose: To evaluate the dose-time dependences of structural changes occurring in collagen within 24 hours to three months after gamma-irradiation at doses from 2–40 Gy in vivo. Materials and methods: Rats tail tendon was chosen as in vivo model, with its highly ordered collagen structure allowing the changes to be interpreted unambiguously. Macromolecular level (I) was investigated by differential scanning calorimetry (DSC); fibers and bundles level (II) by laser scanning microscopy (LSM), and bulk tissue microstructural level (III) by cross-polarization optical coherence tomography (CP-OCT). Results: For (I), the formation of molecular cross-links and breaks appeared to be a principal mechanism of collagen remodeling, with the cross-links number dependent on radiation dose. Changes on level (II) involved primary, secondary and tertiary bundles splitting in a day and a week after irradiation. Bulk collagen microstructure (III) demonstrated early widening of the interference fringes on CP-OCT images observed to occur in the tendon as result of this splitting. At all three levels, the observed collagen changes demonstrated complete remodeling within ∼ a month following irradiation. Conclusion: The time course and dose dependencies of the observed collagen changes at different levels of its hierarchy further contribute to elucidating the role of connective tissue in the radiotherapy process.

Study of the tissue distribution of potential boron neutron-capture therapy agents based on conjugates of chlorin e6 aminoamide derivatives with boron nanoparticles

Boron neutron-capture therapy of cancer is based on the ability of the 10B isotope to capture thermal neutrons; it is one of the most promising techniques in radiation therapy. The high content and selective accumulation of 10B in the tumor tissue are the most important prerequisites for its efficacy. The purpose of this study was to determine the biodistribution of cobalt bis(dicarbollide) conjugates with chlorin e6 amino amide derivatives. Experiments were carried out in Balb/c mice with transplanted CT-26 murine colon carcinoma. Boron-containing conjugates were injected into the tail vein at a dose of 10 mg/kg. The conjugate accumulation in tumor tissue and organs was studied by laser scanning microscopy. Excitation was performed at the wavelength of 514 nm; the signals were recorded in the range of 560–710 nm in increments of 10 nm. To evaluate the amount of the boron conjugate, we calculated the intensity of the fluorescence signal of the samples under investigation. At 3 h after administration of the agent, a high level of fluorescence was observed in the liver, spleen, and lung. The tumor/muscle accumulation ratio was approximately 3. The study demonstrated that boron derivatives of chlorin e6 are promising agents for boron neutron-capture therapy.

Hydrogen peroxide detection in viable and apoptotic tumor cells under action of cisplatin and bleomycin

Abstract Objective: A flow cytometric approach is proposed to assess the hydrogen peroxide (H2O2) level under chemotherapy action separately in viable and apoptotic tumor cells. Materials and methods: For studying the involvement of H2O2 in the process of cell death, the genetically encoded fluorescent sensor HyPer2, apoptosis marker PE Annexin V and vital dye 7-AAD were employed. The approach was used for testing the capacity of two cytotoxic drugs, cisplatin and bleomycin, to change the intracellular H2O2 concentration, depending on the stage of cell death. Results: An increase in HyPer2 fluorescence has been revealed in cells undergoing apoptosis under cisplatin action. This finding indicates that accumulation of H2O2 accompanies the cisplatin-induced apoptotic reaction. HyPer2 response was also revealed in negative to PE Annexin V viable cells which can be explained either by participation of H2O2 in the earliest stages of apoptosis or in a cell response to a non-fatal injury. Under bleomycin action, neither an apoptotic reaction nor changes of fluorescence intensity HyPer2 were detected, allowing one to assume that H2O2 is not involved in the reaction of tumor cells to bleomycin. Conclusion: The proposed approach can be used for studying the mechanisms of cell death under action of any types of antitumor drugs.

OCT visualization of mucosal radiation damage in patients with head and neck cancer. Pilot study

We present the results in optical coherence tomography (OCT) visualization of structural changes in human oral cavity and pharyngeal mucosa for 14 patients in the process of radiation and chemoradiation therapy. Typical mucosal changes are seen as a decrease in the tissue layer contrast progressing to a complete contrast loss as severe mucosal damage occurs. Similar evolution in OCT images was observed for all patients. OCT changes can be seen prior to visual mucosal changes and increased as more mucosal damage occurs. Moreover, OCT was able to obtain information on the specific features of the patient response depending on the irradiation method and the individual radiosensitivity.

OCT visualization of acute radiation mucositis: pilot study

We present pilot results in optical coherence tomography (OCT) visualization of normal mucosa radiation damage. 15 patients undergoing radiation treatment of head and neck cancer were enrolled. OCT was used to monitor the mucositis development during and after treatment. OCT can see stages of radiation mucositis development, including hidden ones, before any clinical manifestations.

In-vivo longitudinal imaging of microvascular changes in irradiated oral mucosa of radiotherapy cancer patients using optical coherence tomography

Mucositis is the limiting toxicity of radio(chemo)therapy of head and neck cancer. Diagnostics, prophylaxis and correction of this condition demand new accurate and objective approaches. Here we report on an in vivo longitudinal monitoring of the oral mucosa dynamics in 25 patients during the course of radiotherapy of oropharyngeal and nasopharyngeal cancer using multifunctional optical coherence tomography (OCT). A spectral domain OCT system with a specially-designed oral imaging probe was used. Microvasculature visualization was based on temporal speckle variations of the full complex signal evaluated by high-pass filtering of 3D data along the slow scan axis. Angiographic image quantification demonstrated an increase of the vascular density and total length of capillary-like-vessels before visual signs or clinical symptoms of mucositis occur. Especially significant microvascular changes compared to their initial levels occurred when grade two and three mucositis developed. Further, microvascular reaction was seen to be dose-level dependent. OCT monitoring in radiotherapy offers a non-invasive, convenient, label-free quantifiable structural and functional volumetric imaging method suitable for longitudinal human patient studies, furnishing fundamental radiobiological insights and potentially providing useful feedback data to enable adaptive radiotherapy (ART).

The study of hydrogen peroxide level under cisplatin action using genetically encoded sensor hyper

The aim of the work was to study the participation of hydrogen peroxide in reaction of cervical cancer cell line HeLa Kyoto on cisplatin action. Determination of hydrogen peroxide level was performed using genetically encoded fluorescent sensor HyPer2. The dependence of cell viability on cisplatin concentration was determined using MTT assay. Mechanisms of cell death as well as HyPer2 reaction was revealed by flow cytometry after 6-hours of incubation with cisplatin in different concentrations. Cisplatin used in low concentrations had no effect on hydrogen peroxide level in HeLa Kyoto cells. Increase of HyPer2 fluorescence was detected only after exposure with cisplatin in high concentration. The reaction was not the consequence of cell death.

Comparative study of tumor hypoxia by diffuse optical spectroscopy and immunohistochemistry in two tumor models

The capabilities of diffuse optical spectroscopy for noninvasive assessing of oxygen status in experimental tumors have been demonstrated. Specific features of the distribution of total hemoglobin, oxygenated hemoglobin, deoxygenated hemoglobin, and blood-oxygen saturation were shown on two tumor models having different histological structure and functional characteristics. The results obtained by the optical technique were verified by immunohistochemical study of tissue samples marked with exogenous marker of hypoxia--pimonidazole.

The study of radiation-induced damage and remodeling of extracellular matrix of rectum and bladder by second-harmonic generation microscopy

Adverse events in normal tissues after irradiation of malignant tumors are of great importance in modern radiation oncology. Second harmonic generation (SHG) microscopy allows observe the structure of collagen fibers and bundles without additional staining. The study objective was evaluation the dose-time dependences of the structural changes occurring in collagen of rat rectum and bladder after gamma-irradiation. Animals were irradiated by a local field at single doses of 10 Gy and 40 Gy. The study of collagen state was carried out in a week and a month after radiation exposure. Paraffin-embedded material was sectioned on the slices 10 mkm thick and SHG-imaging was performed by LSM 510 Meta (Carl Zeiss, Germany). Excitation was implemented with a pulsed (100-fs) titanium-sapphire laser at a wavelength of 800 nm and a pulse repetition frequency of 80 MHz, registration was performed at two wavelengths: 362-415 nm according to collagen fluorescence and 512-576 nm according to myoglobin fluorescence. In a week after irradiation, sings of epithelial damage and edema of submucosal layer, more significant after the dose of 40 Gy were observed on LSM-images. The SHG signal decreased at this time reflecting the processes of collagen degradation independently either in bladder or in rectum. In a month after radiation the increase of size and number of collagen-bearing structures was observed, more essential after irradiation in a dose of 40 Gy. LSM microscopy with SHG allows evaluate changes of normal tissues after ionizing radiation and get information in addition to standard and special histological staining.

Multicolor frequency-domain diffuse optical tomography for detection of breast cancer

Diffuse Optical Tomography (DOT) is based on acquiring information from multiply scattered light which penetrates into the tissue up to depths of several centimeters. This technique allows for imaging of absorbing and scattering inclusions inside tissue and distinguishing between them after computer processing of an image. An experimental setup for multicolor frequency-domain diffuse optical tomography (FD DOT) to visualize neoplasia of breast tissue and to estimate its size has been created. A breast is scanned in the transilluminative configuration by a single source and detector pair. Illumination at three wavelengths (684 nm, 794 nm, and 850 nm) which correspond to different parts of the absorption spectrum provides information about concentration of the main absorbers (oxygenated hemoglobin, deoxygenated hemoglobin, and fat/water). Source amplitude modulation at 140 MHz increases spatial resolution and provides separate reconstruction of scattering and absorption coefficients. In vivo study of breast carcinoma has been performed. Maps of 2D distributions of reconstructed absorption and scattering coefficients and concentration of hemoglobin have been obtained. An increase of absorption and scattering coefficient, total hemoglobin concentration and decrease of blood oxygen saturation is observed in the tumor area in comparison with the surrounding tissue. We can conclude that FD DOT technique confirms a possibility of detecting neoplastic changes.