Skull Optical Clearing Window For Cortical Imaging

In the past decades,since the development of transgenic technology,two-photon microscopy has made it possible to image neurons,glia,and microvasculature in live mice,over time intervals from seconds to years.This technique has become a powerful tool to help understand how the brain works.However,the strong scattering caused by the skull over the cortex limits the penetration depth of light in tissues,and thus hinders the observation of fluorescently labeled neuronal structures and microvasculature.To overcome this obstacle,various cranial window methods were developed.But these methods present limitations.The tissue optical clearing technique can reduce the scattering of tissue,which provides a new way to solve the problem.However,the optical clearing method is widely used in the ex vivo studies of various tissues and organs,and these are few studies on that how to make the living tissues transparent.This study is aimed to develop an innovative cranial window based on the tissue optical clearing technique(named as SOCW),which can be used to clear the mouse skull in vivo very quickly.Combined with two-photon microscopy,this technique allows imaging of the cortical neurons,glia and vasculature at cellular / subcellular resolution.(1)The screening of skull optical clearing agents and the establishment of the clearing method: based on the special layered structure and composition of skull tissue,the reagents were selected after performing the ex vivo and in vivo experiments,and finally the skull optical clearing agents(SOCAs)include collagenase,EDTA disodium salt and glycerol.And different optical clearing methods were developed for mice of different ages.(2)The performance of SOCW for cortical imaging: By combining the SOCW with the two-photon microscopy,we evaluate the validity,reproducibility,compatibility of the SOCW.The results show that the image contrast,the signal to noise ratio,and the imaging depth enhanced significantly after treatment with SOCAs.Through the SOCW,researchers can repeatedly image the neurons,glia,and vascular in the superficial layers of the cortex(250 μm).(3)The safety assessment of SOCW: We performed both in vivo and ex vivo experiments to assess the response of microglia,the expression of glial fibrillary acidic protein(GFAP),and the blood flow/vascular remodeling to assess the safety of SOCW.The microglia,astrocytes and vascular data collectively show that this method induces neither an activation of microglia nor increased expression of GFAP,induces neither the change in blood flow nor vascular remodeling.Whereas,we can conclude that the technique is safe and reliable.(4)The applications of SOCW: Firstly,we monitored the dynamics in dendrites and microglia after two-photon laser ablation,the dendrites in laser injury side formed bead-like structures,while the sites that did not suffer damage remained in a normal state.Microglia soma did not show any significant movement,but the processes with bulbous termini immediately moved towards the site of injury.Then we monitored changes in cortical dendritic spines and microglia after vascular infarct.The results show that the dendrites formed bead-like structures near the infarcted area,whereas the microglia remained in normal state,but at 24 hours after infarct,the morphology and distribution of microglia completely changed.Finally,we applied this technique to study the postsynaptic dendritic spine plasticity in the critical period,the results show that spine formation and elimination were 3.20% ± 0.21% and 1.83% ± 0.52%,respectively.Compared with dendritic spines,filopodia exhibited higher motility.And filopodia can even convert into spine-like protrusions,denmonstrating that the filopodia are likely to be the precursors of dendritic spines.These results demonstrate the dynamic nature of these protrusions,which indicate that the plasticity of dendritic protrusions during the third week is very intense.We develop an innovative cranial window based on the tissue optical clearing,and the method is realized by applying the optical clearing agents to the bare skulls(hair and skin removed)of living mice,and thus forming a visible window of the cortex.Combined with two-photon microscopy,this technique allow us to repeatedly image neurons,glia,and cerebral vasculature in the cortical layer with high resolution.Given its simplicity,safety,and excellent performance,this technique holds great promise for the studies of neural/vascular structure and function in physiological or disease states.