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

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 disorganization 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.

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Kuznetsov S.S., Dudenkova V.V., Kochueva M.V., Kiseleva E.B., Ignatieva N.Yu., Zakharkina O.L., Sergeeva E.A., Babak K.V., Maslennikova А.V. ​Multiphoton Microscopy in the Study of Morphological Characteristics of Radiation-Induced Injuries of the Bladder. Sovremennye tehnologii v medicine 2016; 8(2): 31, https://doi.org/10.17691/stm2016.8.2.04