| Glioma is the most common primary tumors in the central nervous system,accounting for more than 80%of the malignant tumors in the adult central nervous system.Of those,glioblastoma multiform(GBM)is the most common and the most malignant.GBM is usually composed of poorly differentiated,pleomorphic tumor cells with nuclear atypia and brisk mitotic activity,glomeruli-like microvascular proliferation and palisading necrosis.At present,the most widely accepted treatment for GBM is surgical resection within in the maximum safety,combined with radiotherapy and chemotherapy.But the prognosis of GBM patients is not optimistic.The heterogeneity of GBM,with the deepening of the understanding,is considered to be the main cause of various treatment resistance and poor prognosis.However,traditional two-dimensional(2D)methods,such as Hematoxylin-Eosin(HE)staining,Immunohistochemistry(IHC)and Immunofluorescence(IF),are difficult to fully reflect the spatial heterogeneity of GBM.In recent,optical tissue clearing method,which uses chemical reagents to make organs or bulk tissues clearing,combined with three-dimensional(3D)imaging technology simultaneously has been widely used in the research of neural networks.In this study,we compared the presentation methods of image information of 3D imaging technology with the 2D imaging technology from four aspects:cell morphology,cell distribution characteristics,tissue structure,spatial distribution heterogeneity of GBM cells.Chapter 1 Optical tissue clearing methods are applicable to normal brain tissue and GBM tissueMethod:CUBIC and iDISCO+optical tissue clearing methods were used to treat the normal rat brain,human GBM and normal brain tissues respectively,and DAPI was used to stain the nucleus.Then multi-photon microscopy was used to evaluate the result of transparency 3D imaging.Result:After using CUBIC and iDISCO+optical tissue clearing methods to treat the rat brain and GBM tissue respectively,it was observed that the tissue was transparent and the light penetrated was not distort obviously.The multi-photon microscope can clearly and detailedly image human GBM and normal brain tissue from different angles and depths.Chapter 2 Comparison of 2D and 3D imaging methods in brain tissue and GBM tissueMethod:IHC and IF were applied to the paraffin embedded sections and GBM primary cell for staining and microscopic imaging;using optical tissue clearing methods to make brain transparent,staining marker and multi-photon microscope 3D imaging.Result:Compared with cell morphology,distribution and large-scale space,it is found that the images are clear in 2D,but observation in 3D is not available,and there is no three-dimensional shape.Qualitatively and quantitatively,3D image can show the cell morphology and distribution from different angles,depths and layer.It also clearly showing the myelin sheath and blood vessels in brain tissue,It shows the advantages of 3D imaging to reflect the construction of tissue.Chapter 3 3D imaging provides accurate spatial heterogeneity information for GBMMethod:Using optical tissue clearing methods and IF to make GBM tissue clearing,cell stemness,astrocyte differentiation,vessel and immune cell were stained,analyzed with multi-photon microscopy and fluorescence microscope respectively.Result:The density of tumor cells,microvascular and immune cells in GBM tissue is uneven.In general,2D imaging method accurately shows the cell shape in plane and protein expression location.3D imaging method shows an overwhelming advantage in the cell three-dimensional shape,distribution and large-scale tissue structure across large-scale space. |
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