To Assess The Diabetes-induced Changes In Skin Structure And Vascular Function Of Mice By Tissue Optical Clearing Imaging

Diabetes,as a chronic metabolic disease,can induce the vascular dysfunction,even cause changes in composition and structure of tissue,such as skin.In fact,the changes of skin are the most common clinical manifestations of diabetes mellitus,and can occur in the early stage of diabetes.And skin disorders may also be related to complications of other organs,thus skin may be used as a basis for relevant diagnosis.Skin is an important component of peripheral circulation,and it has accessible and rich microvasculature.Especially,skin,an easy-to-detect target,can be used as an effective research target.Thus,it is very important to evaluate the changes of skin structure and microvascular function,but most of studies are based on in vitro experiments.Optical imaging technology provides a very powerful tool for imaging tissue structure and monitoring vascular function.However,the high scattering characteristics of tissues greatly limit the light penetration in tissues,and also limit the performance of optical imaging technology.Recently,tissue optical clearing technique fundamentally can overcome the high scattering of turbid tissue,and effectively improve the quality of in vivo optical imaging.It hopefully provides an effective method for in vivo assessment of skin structure and vascular function changes caused by diabetes mellitus.This work focuses on the above problems.The main contents of this work are as follows:(1)Study on the in vivo skin optical clearing method: Based on theoretical simulation and experiments,the skin optical clearing potential of chain sugar alcohol molecule and ring sugar molecule was compared and analyzed.It was found that cyclic sugars with larger molecular effective coverage surface area had better skin optical clearing potential.Therefore,the skin optical clearing ability of monosaccharides and disaccharides was further compared and analyzed to find reagents with better optical clearing efficiency.The results showed that sugar had better optical clearing potential compared with the alcohol,and disaccharides had better skin optical clearing potential than monosaccharides,in which sucrose was a best skin optical clearing agents.Therefore,a skin optical clearing method based on the sucrose was established.(2)Study on the lookup-table-based hyperspectral blood oxygen evaluation method: A lookup-table-based hyperspectral blood oxygen evaluation method was developed for hyperspectral imaging.And combining with the developed skin optical clearing method,the skin microvascular oxygen response during hypoxic stimulation was dynamically monitored with high resolution.Compared to the common blood oxygen evaluation methods,this blood oxygen evaluation method has a good accuracy in determining blood oxygen,and it can take into consideration of the complex tissue structure and optical properties.(3)To assess type 1 diabetes induced skin vascular dysfunction in vivo: With the assistance of newly-developed in vivo tissue optical clearing techniques,we dynamically monitored the responses of cutaneous vascular blood flow and blood oxygen with the development of diabetes by combing laser speckle contrast imaging with hyperspectral imaging.It can be found that the abnormal changes in cutaneous microvascular blood oxygen response caused by diabetes was earlier than that of blood flow response.Further,compared with the abnormal response of cerebral vascular function caused by diabetes,it was found that the abnormal response of cortical blood flow and blood oxygen occurred almost simultaneously with the abnormal response of skin blood oxygen.Additionally,the causes of vascular abnormal responses were also explored,including the levels of vascular myosin light chain phosphorylation and the glycated hemoglobin.(4)Quantitative assessment of diabetes-induced changes in skin structure and vascular permeability: Evans blue dye as the indicator,and with the assistance of optical clearing skin window,a method was established for in vivo quantitative monitoring of vascular permeability.The changes of skin vascular permeability in diabetic and normal mice were dynamically monitored,it can be found that increase of skin vascular permeability induced by diabetes.Additionally,we used two-photon imaging to realize in vivo 3D visualization of skin,and quantitatively evaluated the changes in skin collagen structure by texture analysis based on analysis of second harmonic generation images in diabetic mice.The results showed that comparing to the normal mice,the integrated two-photon autofluorescence intensity of the diabetic mice was significantly increased but the integrated second harmonic generation intensity was significantly decreased.The results are consistent with the histological analysis,it indicates the changes of collagen distribution can be quantitatively evaluated by texture analysis method.The tissue optical clearing imaging method developed in this study provides an important tool for in vivo monitoring skin structure and vascular functional response.It is very important to in vivo assess vascular dysfunction with the development of diabetes.That is very helpful to better understand the microvascular dysfunction,and has valuable reference for the diagnosis and treatment evaluation of medical intervention towards diabetes.