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2013/2014 ; The main aim of this project is to provide an answer to the following issues: - on the one hand the definition of the cellular and molecular mechanisms of action of the laser therapy and its interaction with tissues - on the other hand the safety of laser therapy and is potential consequences on cancer behaviour We created a mouse model of oral carcinogenesis to assess potential differences in tumour angiogenesis in tissues treated with laser therapy compared to the control ones, by using both histological analysis and injection of Nano FluoSpheres®. A chemical carcinogen (4-NQO) dissolved in their drinking water was administered to C57BL/6 female mice (n = 50), 8-week old, since this compound is able to induce the formation of multiple oral tumours. Among these, 25 mice underwent to 4 session of laser therapy on consecutive days employing the HPLT-1 protocol, while the remaining mice were used as controls. During the 21st week, 15 animals per group were sacrificed to perform an accurate histological analysis of their tongue, while 10 were subjected to a quantitative assessment of angiogenesis through a 3D reconstruction of the tumour vascular network after the in vivo perfusion with Nano FluoSpheres®. Any increase concerning neither the number/extension of dysplastic and neoplastic areas nor tumour angiogenesis was registered in the treated group. Moreover, treated animals showed a tendency to border and to isolate tumour areas. The laser seemed to normalize tumour vessels, promoting their covering by smooth muscle cells, thus reducing ectasia and vascular permeability, as assessed by reduced Nano FluoSpheres® leakiness. The histological analysis performed on the oral carcinogenesis mice model was compared to the images of the same tumours acquired by Narrow Band Imaging. Three raters experienced in the use of this technology analyzed the images, classifying all visible lesions according to different pathological grades; the obtained results were than compared with the histological analysis, used as reference standard. The statistical analysis revealed both high sensitivity (96%) and specificity (99%) for this technology. Supported by other studies, the Narrow Band Imaging is expected to hold great potential for the clinical evaluation of tumour angiogenesis, as well as for the early detection of potentially malignant lesions of the oral cavity. The important clinical outcome in term of wound healing and our interest in the analysis of cell behaviour after laser therapy were the starting point for the evaluation of the effect of laser therapy on different cell lines: Human Skin Fibroblasts, Human Umbilical Vein Endothelial Cells, Human Coronary Artery Smooth Muscle Cells, Neonatal Rat Ventricular Myocytes, Human Bone Osteosarcoma Epithelial Cells and Mouse B16F10 Melanoma Cells. We set different powers, energies and wavelengths, and we performed the evaluations at different experimental times (6, 24, 48 and 96 hours after the irradiation). In general, laser irradiation resulted in an increase of both cell metabolism (ATPlite) and proliferation (cell count, AlamarBlu, BrdU incorporation), albeit with different timing and intensity in the various cell lines. Consistent with published results, we observed a clear increase in cancer cell metabolism upon laser irradiation; to evaluate the cancer behaviour in vivo, the same melanoma cells were implanted in C57BL/6 female mice (n = 16), 6-week old, at the dorsal subcutaneous level. As soon as the masses were visible to the naked eye (approximately on day 10), mice were homogeneously divided into 4 groups according to tumour size: 3 groups were subjected to different laser protocols (LPLT-6; MPLT-13 and HPLT-7) for 4 consecutive days (days 11 to 14), while the fourth group was used as control. On day 15, all animals were euthanized to measure the tumour volume and weight. A deep histological analysis on tumour invasion and cancer immune response (CD1a, CD4, CD8, CD25, CD68 kp1 and Melan-A) was performed, as well as the analysis of the expression levels of cytokines involved in the immune system activation (TNFα, IFNα and IFNγ). Laser therapy did not foster tumour growth or invasiveness (CD68 kp1 and Melan-A), but rather seemed to contain its extension. Moreover, in the laser groups, tumour infiltration by immune cells was much more higher compared to the control ones (CD4+, CD8+, CD25+ cells), consistent with the increased expression of IFNγ. Of notice, CD1a positive dendritic cells were particularly abundant in the dermis in the control group, while they migrated to wrap" the tumour in laser groups. Based on these results, we applied the same laser protocols on primary mouse bone marrow dendritic cells, with and without lipopolysaccharide stimulation. These cells did not enhance cell metabolism upon laser treatment, but reduced TNFα and increased IFNγ expression. Finally, CD-1 female mice (n = 30), age 6-7 weeks old, were used to assess the expression of different cytokines (Collagen I, Collagen III, Collagen IV, FSP1, IL-2, IL-6, IL-10, IFNα, IFNβ, IFNγ, MMP-9, PDGFβ, TGFβ, TNFα) after the laser therapy at the dorsal level with and without the presence of a skin wound. The analysis confirmed an increase in the IFNγ expression
Note:
Dissertation Università degli studi di Trieste 2015
Language:
English
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