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.
Radiation therapy, widely used in the treatment of a variety of malignancies in the pelvic area, is associated with inevitable damage to the surrounding healthy tissues. We have applied atomic force microscopy (AFM) to track the early damaging effects of ionizing radiation on the collagen structures in the experimental animals' bladder and rectum. The first signs of the low-dose radiation (2 Gy) effect were detected by AFM as early as 1 week postirradiation. The observed changes were consistent with initial radiation destruction of the protein matrix. The alterations in the collagen fibers' packing 1 month postirradiation were indicative of the onset of fibrotic processes. The destructive effect of higher radiation doses was probed 1 day posttreatment. The severity of the radiation damage was proportional to the dose, from relatively minor changes in the collagen packing at 8 Gy to the growing collagen matrix destruction at higher doses and complete three-dimensional collagen network restructuring towards fibrotic-type architecture at the dose of 22 Gy. The AFM study appeared superior to the optical microscopy-based studies in its sensitivity to early radiation damage of tissues, providing valuable additional information on the onset and development of the collagen matrix destruction and remodeling.
The objective of the study is the quantitative analysis of the dose-time dependences of changes occurring in collagen of bladder and rectum after gamma-irradiation using optical methods [nonlinear microscopy in a second harmonic generation (SHG) detection regime and cross-polarization optical coherence tomography (CP OCT)]. For quantitative assessment of the collagen structure, regions of interest on the SHG-images of two-dimensional (2-D) distribution of SHG signal intensity of collagen were chosen in the submucosa. The mean SHG signal intensity and its standard deviation were calculated by ImageJ 1.39p (NIH). For quantitative analysis of CP OCT data, an integral depolarization factor (IDF) was calculated. Quantitative calculation of the SHG signal intensity and the IDF can provide additional information about the processes of the collagen radiation-induced degradation and subsequent remodeling. High positive correlation between the mean SHG signal intensity and the mean IDF of bladder and rectum demonstrates that CP OCT can be used as an "optical biopsy" in the grading of collagen radiation damage.
The aim of the investigation was to assess the feasibility of multiphoton microscopy (MPM) for studying dynamics of bladder structural changes following a single exposure to gamma-radiation at various doses (2, 10, and 40 Gy) in experiment.Materials and Methods. Specimens of rat bladders after a single local radiation at the dose of 2, 10, and 40 Gy were the objects of investigation (9 groups with two rats for each dose and term, and two intact rats -20 observations in all). The study was carried out 1 day, 1 week, and 1 month after radiation exposure. Part of the histological bladder preparations was stained with picrofuchsin according to Van Gieson method. The other part of the sections, obtained from the same blocks, was investigated using MPM without additional staining. For this purpose a laser scanning microscope LSM Axiovert 510 Meta (Carl Zeiss, Germany) was used. Excitation was generated with a femtosecond Ti:Sapphire laser (MAI TAI HP, Spectra Physics, USA) at the wavelength of 800 nm, registration was performed in the range of 362-415 nm (second harmonic signal from collagen) and 512-576 nm (signal of two-photon excited elastin autofluorescence).Results. Application of MPM method allowed us to find out, that in early terms (1 day and 1 week) after radiation exposure the process of alteration of collagen-containing structures of bladder walls was a leading one at all selected doses. A month after 2 and 10 Gy radiation increase in collagen structures was registered, speaking of the onset of radiation fibrosis formation. At a dose of 40 Gy decrease of second harmonic signal retained in the extracellular matrix of the bladder wall. It allowed us to draw a conclusion on a long-term disorganization of collagen at high radiation doses.Conclusion. MPM method makes it possible to estimate, that structural destruction of extracellular tissue matrix occurs even after low radiation doses and in early terms after radiation exposure, which is not possible to reveal using standard microscopy. Duration of BiomedicAl investigAtionsdisorganization process of collagen-containing structures depends on the radiation dose: high doses result in longer-lasting alterations. MPM enables also the assessment of the course of restorative processes.
The study aimed to assess the characteristics of the urinary bladder extracellular matrix after radiotherapy for cervical cancer and hysterocarcinoma using nonlinear microscopy.Materials and Methods. Two groups of patients were studied. The first group (n=75) involved female patients with severe complications after combined radiotherapy for cervix cancer or endometrial cancer. Adverse events of urinary bladder developed within the period from a year to eleven years. The second group (n=80) consisted of female patients suffering from chronic cystitis of bacterial etiology, their past history being over 3 years. We carried out a comparative analysis of the cystoscopic pattern of mucosa and the morphological analysis findings of urinary bladder bioptates.For the first time there was studied the extracellular matrix state of the bladder connective tissue after radiotherapy by nonlinear microscopy carried out in the modes: second harmonic generation and two-photon excited autofluorescence to examine the state of collagen and elastin, respectively. To verify the obtained images we studied parallel histological sections stained by hematoxylin and eosin and picrosirius red.Results. Nonlinear microscopy in radiation and chronic cystitis revealed similar inflammatory changes and tissue fibrosis. The intensity of radiation changes of the urinary bladder tissues depended directly on time after radiation, they being more intense and gross compared to those in chronic cystitis. Nonlinear microscopy enabled to reveal the difference in collagen and elastin structures after urinary bladder radiation damage of various severity. The structure of collagen fibers in II severity degree of radiation was preserved, the fibers being more packed, while III degree was characterized by marked disarrangement of collagen fibers.Conclusion. The combination of optical methods (nonlinear microscopy combined with specific staining of histological preparations) enables to assess objectively structural changes of the urinary bladder extracellular matrix and determine the intensity of alterations after ionizing radiation.The findings can serve as the basis to develop the approaches to visual and quantitative evaluation of the results of noninvasive optical techniques (e.g., polarization modifications of optical coherence tomography) to monitor radiation-induced damage in the urinary bladder.
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