Optical Clearing Imaging For Investigating Alternations Of Subcutaneous Vascular And Monocytic Motility Function In Mice Model Of Diabetes

Diabetes not only results in abnormal subcutaneous vascular structure and function,but also induces dysfunction of the immune response of immunocytes(e.g.monocytes).But the investigations about how subcutaneous vascular function and monocytic immune function change with the development of diabetes have rarely been reported,the primary reason is the lack of the relevant research method to monitor this dynamic changes.Optical imaging technologies can provide vascular and cellular structure and function with the incomparable imaging resolution and contrast that other methods cannot achieve.But the turbid characteristic of skin induces the dramatically decreased imaging resolution and contrast in the internal deeper tissue.Skin optical clearing technique is expected to provide a promising solution for this problem,the combination of skin optical clearing technique and diverse optical imaging modalities will provide an important tool for tracing the dynamic changes of subcutaneous vascular function and monocytic motility function with the development of diabetes.This thesis focused on how the subcutaneous vascular function and monocytic motility function changed with the development of diabetes.In order to achieve this research goal mentioned above,we developed dorsal and footpad skin optical clearing respectively to improve the qualities of blood flow and cell imaging,which were further combined with diverse optical imaging,namely skin optical clearing imaging we called,to investigate progressive diabetes-induced effects on vascular function and monocytic motility function,and obtained some meaningful results.The main contents and results were as follow:(1)Study of skin optical clearing method for improving the qualities of blood flow and cell imaging:considering the limitations of pre-existing screening approaches for skin optical clearing agents,we further investigated the screening approach of skin optical clearing agents.Based on this proposed screening approach and the distinctive features of dorsal and footpad skin,the optical clearing methods that can dramatically improve the performances of blood flow and cell imaging were established respectively,theirs effectiveness and safety were also investigated.Results demonstrated that optical clearing agents-induced in vivo skin optical clearing efficacy positively correlated with the optical clearing potential and the penetration enhancing effect of reagents contained,the established dorsal and footpad skin optical clearing methods can not only significantly ameliorate subcutaneous blood flow and cell imaging,but also had no influences on blood vessels and cells,which illustrated its safety.(2)The effects of progressive diabetes on subcutaneous vascular function:the established skin optical clearing method was combined with laser speckle contrast imaging,hyperspectral imaging and confocal microscopy to investigate the effects of progressive type 1 diabetes(T1D)on blood flow dynamics and vascular permeability.Results demonstrated that the responses of blood flow and oxygen saturation to norepinephrine were consistent with its pharmacological function under condition of physiological status.Diabetic pathological status induced much more decreased blood flow velocity and oxygen saturation in subcutaneous blood vessels(especially the veins),and this changing tendency became much more significant as progressive T1D developed(especially at T1D-2w and TlD-4w).The expression level of adrenal receptor and the vascular permeability increased significantly with the development of T1D.(3)The effects of progressive diabetes on monocytic motility function:the established skin optical clearing method was combined with confocal microscopy to investigate the effects of progressive T1D on monocytic recruitment and its motility at different depths(e.g.40μm and 90μm)during inflammation(e.g.DTH,delayed-type hypersensitivity).Results demonstrated that progressive T1D(especially at T1D-2w and TlD-4w)significantly increased the extent of monocytic recruitment but attenuated its motion displacement and motion velocity during DTH,which indicated that the immune function of monocytes was gradually impaired as progressive T1D developed.The motion parameters of monocytes at different depths around DTH foci were indeed different,the motion ranges at shallow and deep tissue were both attenuated,and the differences of motion ranges between different depths were also gradually shrank as progressive T1D developed.In summary,the changes of subcutaneous vascular function and monocytic motility function with the development of T1D were investigated in this thesis via skin optical clearing imaging,the results obtained provide some important basises for describing the changing tendency of vascular function and immunocytic immune function as progressive T1D develops,it will provide some valuable references for diabetic clinical interventional therapy strategies and efficacy evaluation in the future.