An Optical Clearing Skull Window For Cerebrovascular Imaging And Manipulation

With the rapid development of optical imaging technologies,it can be realized to monitor the structural and functional changes of cortical vessels and glial cells in physiopathological conditions,as well as laser manipulation of cortex for further therapy.However,the turbid skull above the cortex severely affects the light penetration in biological tissues,therefore,it has great limitations to the application of optical techniques in in vivo cortial imaging and manipulation.Researchers often partly or completely remove the skull to establish the surgical cranial windows for dynamically monitoring the cerebrovascular structure and function with high resolutions.But all of these cranial windows have some deficiencies,such as cortical inflammations,low repeatability and complex surgical operations.Tissue optical clearing technique provides us with a new idea.Though some optical clearing skull windows have been proposed,they still have some restrictions,including poor transparency,low repeatability and limited application for infantile mice.This study focuses on in vivo skull optical clearing technique,aiming at developing a more efficient,widely-used and safe optical clearing skull window that not only permits long-term tracing the cerebrovascular structure and function,but also opening blood brain barrier with the assist of photodynamic therapy.(1)The establishment of innovative skull optical clearing method: According to the structure and composition of mouse skull,we carried out in vitro and in vivo assessments to selecte and optimize the best skull optical clearing agent,which consisted of saturated solution of 75% ethyl alcohol and urea as well as sodium dodecyl benzene sulfonate solution.Then,we found that the effect of skull optical clearing was reversible,and the skull could recover to the initial state by saline treatment.Also,we tested its safety for usage,and the results suggested that this method would not induced any cortical inflammation and metabolic toxicity.(2)Monitoring cerebrovascular structure and function through the optical clearing skull window: By employing two-photon imaging,we observed cortical vascular structure with high resolution.The result suggested that the minimum resolvable vascular diameter was 4.5 ?m and the imaging depth reached to 300 ?m after establishing optical clearing skull window.With the help of laser speckle contrast/hyperspectral dual-mode imaging system,we realized bi-hemispheric imaging and monitored the dynamic changes in arteriovenous blood oxygen and blood flow after implementing middle cerebral artery occlusion.In addition,this method was applicable to 2-8 months old mice.On this basis,we firstly realized long-term tracing cerebrovascular structure and function with high resolution.The results suggested that the cortical vessels of adult mouse kept stable during its development,with only a few angiogenesis and development.(3)Opening of blood brain barrier by combining the optical clearing skull window with photodynamic effect: We firstly realized noninvasive blood brain barrier opening by combining photodynamic effect with optical clearing skull window.We found that the blood brain barrier structure changed after photodynamic treatment,in which the basal membrane was intact but the distribution of endothelial cells and astrocytes changed.Furthermore,we qualitatively and quantitively evaluated the differences in photodynamic-induced blood brain barrier opening between infantile and adult mice.The results showed that the young mice were more sensitive than adult mice.In addition,we dynamically monitored the changing process of blood brain barrier permeability and investigated the spatial-temporal variation.The results indicated that blood brain barrier was not instantly opened after photodynamic treatment.Above all,this study mainly proposed an innovative skull optical clearing method of mice.This method features convenience,high-efficiency,switchability,safety and repeatability,which satisfy the requirements for cerebrovascular imaging with high resolution.It will be an important approach to in vivo monitor the occurrence and development of cerebrovascular diseases.Besides,the combination of photodynamic effect with optical clearing skull window was also a powerful tool for the development of drugs for brain diseases.Therefore,this research has great scientific significance and application prospect.