Murine induced colon cancer has been used to demonstrate that Second Harmonic Generation (SHG) microscopy images, combined with Two-Photon Excitation Fluorescence (TPEF) and specific quantization scoring methods allow distinguishing early alterations in colon mucosa. TPEF was used only to identified crypts and submucosa regions, whereas the image analysis was used to get quantitative data (Integrated Intensity and Aspect Ratio scoring) of different cancer stages. The submucosa amount of collagen fibers was significant and their orientation suffering proportional changes with the development of the pathological processes. Both after the fourth and eighth weeks after colon cancer induction, integrated intensity and aspect ratio values have shown significant statistical differences compared with control samples. Thus, SHG microscopy has proved to be a useful quantitative tool to highlight early changes of submucosa and the progression of these through the cancer development.
12 13 BACKGROUND AND AIMS: Colorectal cancer (CRC) is a disease that can be prevented 14 if is diagnosed and treated at pre-invasive stages. Thus, the monitoring of colonic cancer 15 progression can improve the early diagnosis and detection of malignant lesions in the colon. 16 This monitoring should be performed with appropriate image techniques and be accompanied 17 by proper quantification to minimize subjectivity. We have monitored the mice CRC 18 progression by image deconvolution, two-photon emission fluorescence (TPEF) and second 19 harmonic generation (SHG) microscopies and present different quantization indices for 20 diagnosis. METHODS: The Azoxymethane (AOM) / dextran sodium sulfate (DSS) protocol 21 was used. 35 eight-week old male BALB/cCmedc mice were used and distal colon segments 22were dissected at day zero and fourth, eighth, sixteen, and twenty weeks after injection. These 23 segments were observed with linear and nonlinear optical microscopies and several 24 parameters were used for quantification. RESULTS: Crypt diameter higher than 0.08 mm 25 and increased fluorescence signal intensity in linear images; as well as aspect relation above 26 0.7 and altered organization reflexed by high-energy values obtained from SHG images, 27 away from those obtained in normal tissues. CONCLUSION: the combination of linear and 28 nonlinear signals improve the detection and classification of pathological changes in crypt 29 morphology/distribution and collagen fiber structure/arrangement. In combination with 30 standard screening approaches for CRC, the proposed methods improve the detection of the 31 disease in its early stages, thereby increasing the chances of successful treatment. 32 KEYBORDS: colorectal cancer, linear microscopy, nonlinear microscopy, early diagnosis 2 33 34Colorectal cancer (CRC) is a common malignancy, it is the third most common cause of 35 cancer death in both men and women [1]. In general, CRC has a good prognosis, when it is 36 diagnosed and treated at pre-invasive stages [2]. However, the detection of early lesions is 37 still challenging. 38 To systematically characterize early tumoral transformations, an animal model of CRC 39 progression is needed. Mice Azoxymethane (AOM)-induced CRC is one of the most 40 commonly used pre-clinical animal model to mimic human sporadic CRC development [3]. 41 Specifically, the AOM/dextran sodium sulfate (DSS) protocol has been proven a powerful 42 tool for investigating the pathogenesis and chemoprevention of colitis-related colon 43 carcinogenesis [4]. The development of colonic cancer is a multi-stage process [5] and the 44 monitoring of colonic cancer progression can improve the early diagnosis and detection of 45 malignant colon lesions. This examining should be performed with appropriate image 46 techniques and be accompanied by proper quantification to minimize subjectivity. 47 Currently, anatomo-pathological examination is the diagnostic gold standard. The process 48 involves many steps, prior to examination. It is time-consuming and expe...
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