Camila Salata

Up-regulation of angiotensin-converting enzyme and angiotensin II type 1 receptor in irradiated rats

Purpose: To investigate changes in cardiac functional parameters and the cardiac expression of angiotensin-converting enzyme (ACE), angiotensin II type 1 receptor (AT1), procollagen type I (proc-I) and transforming growth factor-β1 (TGF-β1) in rats irradiated at heart. Material and methods: Male Wistar rats were irradiated with a single dose of radiation (0, 5, 10 and 15 Gray [Gy]) delivered directly to the heart and the molecular evaluations were performed at various times post-irradiation (two days, 15 days and four months). The expression of ACE, AT1, proc-I and TGF-β1 were analysed using Real Time-Polymerase Chain Reaction (RT-PCR) and/or Western blotting. Cardiac structural and functional alterations were investigated at the four-month time point by echocardiography and by quantitative methods (stereology). Results: Rats irradiated with 15 Gy showed a modest reduction in the ejection fraction. Cardiac proc-I, TGF-β1, ACE and AT1 were also measurably increased. Conclusions: Irradiated rat hearts show simultaneous elevations in renin-angiotensin system components AT1 and ACE and cardiac remodeling markers proc-I and TGF-β1.

Apoptosis induction of cardiomyocytes and subsequent fibrosis after irradiation and neoadjuvant chemotherapy.

Abstract Purpose: Breast cancer treatments can induce important cardiovascular complications. The aim of this study was to evaluate cardiac alterations after irradiation and chemotherapy in an animal model. Material and methods: Wistar rats were divided into three groups: Control, TC+ IR (received chemotherapy and irradiation) and IR (received only irradiation). After 5 months, echocardiography was performed, the animals were euthanized, and the left ventricle was analyzed using light microscopy techniques and Polymerase Chain Reaction (PCR). Results: Echocardiography showed decreases in ejection fraction and cardiac output, in TC+ IR group. Both TC+ IR and IR showed reduced intramyocardial vessel-to-cardiomyocyte ratio, increased connective tissue, cardiomyocyte hypertrophy, increased numbers of apoptotic nuclei and increased Bax/Bcl2 expression. We also observed increased Transforming growth factor (TGF) beta 1 mRNA expression in both groups, but type 1 Procollagen expression was increased in TC+ IR group only. Conclusions: The study suggests that the induced cardiac remodelling begins with the reduction of intramyocardial vessels in the left ventricle tissue. The main consequence is the loss of cardiomyocytes through apoptosis, leading to the replacement of healthy tissue by fibrous tissue. It was observed that the damage caused by the combination of irradiation and chemotherapy induced functional alterations that did not occur when the animals were only irradiated.

Chemotherapy and radiation regimens to breast cancer treatment induce changes in mRNA levels of renin-angiotensin system related genes in cardiac tissue

Background and aim: Cardiovascular complications are one limitation of breast cancer treatment. The aim of the current study was to investigate whether the renin–angiotensin related genes could be altered by chemotherapy and radiotherapy, using a rat model. Methods: Female rats were divided into three groups: control, chemotherapy + irradiation (TC+IR) and irradiation (IR). Molecular analyses of the left ventricle were performed five months after the end of treatment. The analyses evaluated the changes in mRNA levels of some renin–angiotensin system (RAS) related genes: angiotensinogen, renin, angiotensin-converting enzyme (ACE) and angiotensin II type 1 receptor (AT1) and vascular endothelial growth factor (VEGF), which can be related to ACE production, by RT-PCR. Results: Renin was only observed in treated groups, TC+IR and IR, compared with the control group. ACE and VEGF levels were decreased in TC+IR (p<0.001) and in IR (p<0.001), and AT1 mRNA was higher in groups TC+IR (p<0.01) and IR (p<0.05) compared with the control group. Conclusion: Chemotherapy and irradiation can induce significant changes in some RAS related genes. These alterations are important to understand the pathways and consequences beyond cardiotoxicity induced by breast cancer treatments.

Caspase-3 activation and increased procollagen type I in irradiated hearts

The caspase-3-cleaved presence was evaluated in this study in the heart of irradiated rats, during the decline of ventricular function. Female Wistar rats were irradiated with a single dose of radiation (15 Gy) delivered directly to the heart and the molecular, histological and physiological evaluations were performed at thirteen months post-irradiation. The expressions of procollagen type I, TGF-ß1 and caspase-3-cleaved were analyzed using Western blotting. Cardiac structural and functional alterations were investigated by echocardiography and electron microscopy. In the irradiated group, the levels of procollagen type I, TGF-ß1 and caspase-3-cleaved are increased. Significant histological changes (degeneration of heart tissue and collagen deposition) and functional (reduced ejection fraction) were observed. Data suggest that the cardiac function decline after exposure to ionizing radiation is related, in part, to increased collagen and increased caspase-3-cleaved.

A Feasibility Study of Fricke Dosimetry as an Absorbed Dose to Water Standard for 192Ir HDR Sources

High dose rate brachytherapy (HDR) using 192Ir sources is well accepted as an important treatment option and thus requires an accurate dosimetry standard. However, a dosimetry standard for the direct measurement of the absolute dose to water for this particular source type is currently not available. An improved standard for the absorbed dose to water based on Fricke dosimetry of HDR 192Ir brachytherapy sources is presented in this study. The main goal of this paper is to demonstrate the potential usefulness of the Fricke dosimetry technique for the standardization of the quantity absorbed dose to water for 192Ir sources. A molded, double-walled, spherical vessel for water containing the Fricke solution was constructed based on the Fricke system. The authors measured the absorbed dose to water and compared it with the doses calculated using the AAPM TG-43 report. The overall combined uncertainty associated with the measurements using Fricke dosimetry was 1.4% for k = 1, which is better than the uncertainties reported in previous studies. These results are promising; hence, the use of Fricke dosimetry to measure the absorbed dose to water as a standard for HDR 192Ir may be possible in the future.

Evaluation of rib microstructure in Wistar rats using SR-μCT after radiation therapy simulation for breast cancer

A better understanding of biological interactions that occur after exposure to photon radiation is needed in order to optimize therapeutic regimens and facilitate development and strategies that decrease radiation-induced side effects in humans. In this work, ribs of Wistar rat submitted to radiotherapy simulation were imaged using synchrotron radiation computed microtomography at Elettra Synchrotron Laboratory in Trieste, Italy. Histomorphometric parameters were calculated directly from the 3D microtomographic images and showed significant differences between irradiated and non-irradiated groups.

Effects of soft X-ray radiation damage on paraffin-embedded rat tissues supported on ultralene: a chemical perspective

Radiation damage is an important aspect to be considered when analysing biological samples with X-ray techniques as it can induce chemical and structural changes in the specimens. This work aims to provide new insights into the soft X-ray induced radiation damage of the complete sample, including not only the biological tissue itself but also the substrate and embedding medium, and the tissue fixation procedure. Sample preparation and handling involves an unavoidable interaction with the sample matrix and could play an important role in the radiation-damage mechanism. To understand the influence of sample preparation and handling on radiation damage, the effects of soft X-ray exposure at different doses on ultralene, paraffin and on paraffin-embedded rat tissues were studied using Fourier-transform infrared (FTIR) microspectroscopy and X-ray microscopy. Tissues were preserved with three different commonly used fixatives: formalin, glutaraldehyde and Karnovsky. FTIR results showed that ultralene and paraffin undergo a dose-dependent degradation of their vibrational profiles, consistent with radiation-induced oxidative damage. In addition, formalin fixative has been shown to improve the preservation of the secondary structure of proteins in tissues compared with both glutaraldehyde and Karnovsky fixation. However, conclusive considerations cannot be drawn on the optimal fixation protocol because of the interference introduced by both substrate and embedding medium in the spectral regions specific to tissue lipids, nucleic acids and carbohydrates. Notably, despite the detected alterations affecting the chemical architecture of the sample as a whole, composed of tissue, substrate and embedding medium, the structural morphology of the tissues at the micrometre scale is essentially preserved even at the highest exposure dose.

A combination of stereological methods, biochemistry and electron microscopy for the investigation of drug treatment effects in experimental animals

Some chemotherapeutic agents used for breast cancer (BC) treatment can induce severe side effects in the ovarian tissue. The combination of cyclophosphamide and docetaxel (TC) is widely used for BC treatment; however, its late effects in the ovary are not completely understood. The main purpose of this study was to evaluate the structural and ultrastructural alterations in the ovarian stroma induced by TC treatment. Wistar rats were divided into two groups: a control group and a TC group. They were euthanized 5 months after the end of treatment, and their plasma and ovaries were collected. Important alterations were noted. The serum estradiol level was significantly reduced in the TC group compared with the control group. Additionally, the number of apoptotic nuclei was higher in the TC group. The role of the inflammatory response in the development of ovarian damage was investigated, and we found an increased number of mast cells and increased expression of TNF‐α in the TC group. The involvement of fibrosis was also investigated. The results showed that the TC group had increased expression levels of TGF‐β1, collagen type I (col‐I) and collagen type III (col‐III) compared with the control group. Ultrastructural analysis revealed the presence of collagen fibrils in the treated group and illustrated that the ovarian tissue architecture was more disorganized in this group than in the control group. The results from this study are important in the study of chemotherapy‐induced ovarian failure and provide further insight into the mechanisms involved in the development of this disease.

SU-F-19A-02: Comparison of Absorbed Dose to Water Standards for HDR Ir-192 Brachytherapy Between the LCR, Brazil and NRC, Canada.

PURPOSE To compare absorbed dose to water standards for HDR brachytherapy dosimetry developed by the Radiological Science Laboratory of Rio de Janeiro State University (LCR) and the National Research Council, Canada (NRC). METHODS The two institutions have separately developed absorbed dose standards based on the Fricke dosimetry system. There are important differences between the two standards, including: preparation and read-out of the Fricke solution, irradiation geometry of the Fricke holder in relation to the Ir-192 source, and determination of the G-value to be used at Ir-192 energies. All measurements for both standards were made directly at the NRC laboratory (i.e., no transfer instrument was used) using a single Ir-192 source (microSelectron v2). In addition, the NRC group has established a self-consistent method to determine the G-value for Ir-192, based on an interpolation between G-values obtained at Co-60 and 250kVp X-rays, and this measurement was repeated using the LCR Fricke solution to investigate possible systematic uncertainties. RESULTS G-values for Co-60 and 250 kVp x-rays, obtained using the LCR Fricke system, agreed with the NRC values within 0.5 % and 1 % respectively, indicating that the general assumption of universal G-values is appropriate in this case. The standard uncertainty in the determination of G for Ir-192 is estimated to be 0.6 %. For the comparison of absorbed dose measurements at the reference point for Ir-192 (1 cm depth in water, perpendicular to the seed long-axis), the ratio Dw(NRC)/Dw(LCR) was found to be 1.011 with a combined standard uncertainty of 1.7 %, k=1. CONCLUSION The agreement in the absorbed dose to water values for the LCR and NRC systems is very encouraging. Combined with the lower uncertainty in this approach compared to the present air-kerma approach, these results reaffirm the use of Fricke solution as a potential primary standard for HDR Ir-192 brachytherapy.

Analysis of femur head microstructure in ovariectomized rats

It is well accepted that rat ovariectomy (OVX) is a model of estrogen deficiency. OVX played a very important role in the initiating and developing of osteoporosis and it has been shown to be a major risk factor for the development of postmenopausal osteoporosis in women. In this work we used synchrotron radiation computed microtomography to investigate the skeletal effects in femoral head of female Wistar rats after bilateral ovariectomy surgery. The CT system was set up at the SYRMEP beamline in the synchrotron radiation facility ELETTRA (Trieste, Italy). Micro-CT images provided 3D information on precise trabecular microstructure by the reconstruction of multiple 2D images with almost 2 μm resolution. Our aim was to use histomorphometric analysis to reveal the effect of OVX on the three-dimensional (3D) trabecular bone microarchitecture. Evaluated morphometric parameters were trabecular bone volume-tissue volume ratio (BV/TV), trabecular number (Tb.N), trabecular separation (Tb.Sp) and trabecular thickness (Tb.Th). OVX group presented noticeable reduction in the Tb.N and Tb.Th when compared with control group (P 0.05). Our data may help to gain more insight into the potential mechanism of osteoporotic femoral head fractures.