Late Changes in the Extracellular Matrix of the Bladder after Radiation Therapy for Pelvic Tumors.

Radiation therapy is one of the cardinal approaches in the treatment of malignant tumors of the pelvis. It leads to the development of radiation-induced complications in the normal tissues. Thus, the evaluation of radiation-induced changes in the extracellular matrix of the normal tissue is deemed urgent, since connective tissue stroma degradation plays a crucial role in the development of Grade 3-4 adverse effects (hemorrhage, necrosis, and fistula). Such adverse effects not only drastically reduce the patients' quality of life but can also become life-threatening. The aim of this study is to quantitatively analyze the bladder collagen state in patients who underwent radiation therapy for cervical and endometrial cancer and in patients with chronic bacterial cystitis and compare them to the normal bladder extracellular matrix. MATERIALS AND METHODS: One hundred and five patients with Grade 2-4 of radiation cystitis, 67 patients with bacterial chronic cystitis, and 20 volunteers without bladder pathology were enrolled. Collagen changes were evaluated depending on its hierarchical level: fibrils and fibers level by atomic force microscopy; fibers and bundles level by two-photon microscopy in the second harmonic generation (SHG) mode; general collagen architectonics by cross-polarization optical coherence tomography (CP OCT). RESULTS: The main sign of the radiation-induced damage of collagen fibrils and fibers was the loss of the ordered "basket-weave" packing and a significant increase in the total area of ruptures deeper than 1 µm compared to the intact sample. The numerical analysis of SHG images detected that a decrease in the SHG signal intensity of collagen is correlated with the increase in the grade of radiation cystitis. The OCT signal brightness in cross-polarization images demonstrated a gradual decrease compared to the intact bladder depending on the grade of the adverse event. CONCLUSIONS: The observed correspondence between the extracellular matrix changes at the microscopic level and at the level of the general organ architectonics allows for the consideration of CP OCT as a method of "optical biopsy" in the grading of radiation-induced collagen damage.

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

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.

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

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.

Effects of gamma irradiation on collagen damage and remodeling.

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: Rat's 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.