​Multiphoton Microscopy in the Study of Morphological Characteristics of Radiation-Induced Injuries of the Bladder

Abstract

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.

Early Effects of Ionizing Radiation on the Collagen Hierarchical Structure of Bladder and Rectum Visualized by Atomic Force Microscopy

“…These effects advanced with the dose (Fig. 6, S3), completely destroying the initial characteristic basket-weave pattern of the skin in this area (Kotova et al, 2015).…”

“…In our previous studies (Kotova et al, 2015; Timashev et al, 2016), we have developed an AFM-based approach to tracking pathological alterations of the collagen hierarchical structure in ECM of various tissues, which may be used as a complimentary technique to the traditional histological clinical assessment. In this study, we apply the same approach to probe early negative effects of ionizing radiation on the healthy tissue of pelvic organs (bladder and rectum).…”

Quantitative assessment of radiation-induced changes of bladder and rectum collagen structure using optical methods

“…2). 36 A clear dependence of the SHG signal intensity of the bladder wall collagen on the dose and the time after irradiation was observed after the quantitative assessment of SHG-images. After irradiation at a dose of 2 Gy, the values of the intensity of the SHG signal did not differ from the intact bladder samples irrespective of the term after irradiation.…”

“…1(a)]. 36 No changes in the extracellular matrix were detected during the visual assessment of images of the SHG signal distribution of collagen of the bladder wall after exposure to a dose of 2 Gy irrespective of the time after irradiation (Fig. 2).…”

Atomic Force Microscopy of Tissue Sections Is a Useful Complementary Tool in Biomedical Morphological Studies

“…In spite of the recent development of laser microscopic techniques for the collagen studies, such as laser scanning microscopy and optical coherence tomography [3,4], conventional histological techniques remain by far the most widely used methods of connective tissue investigation both in the experimental and clinical setting. Stained histological sections provide the majority of information about the overall appearance of the collagen matrix, elastic fibers, and cells.…”