Three-dimensional Analysis Of Brain Structure Based On Ultrafast Tissue Clearing Technology

In the study of the nervous system,obtaining the complete neuroanatomical structure is considered fundamental to understanding brain function and neuropsychiatric disorders.Therefore,high-resolution and large-scale mammalian whole-brain mapping is often taken as a priority deployment area in brain projects carried out in countries such as Europe,America,China,and Japan.Tissue clearing technology is a new technology developed in recent years for three-dimensional(3D)imaging of tissues.This technique reduces the attenuation of light of biological tissues by using a variety of different strategies,the whole brain or even the whole body of mice can be completed cleared,and then the fine 3D structure of large-volume samples can be obtained.It provides a powerful methodological tool for studying the structure of biological tissues at the 3D level.Most of the current tissue clearing methods are designed for the clearing of large-volume samples.To complete the clearing of large-volume samples within an acceptable time,relatively severe clearing conditions are used.Current tissue-clearing workflows have several disadvantages,including complex protocols,time-consuming application,fluorescence quenching,and the use of toxic reagents.These problems have affected the widespread application of tissue clearing technology in routine experiments.Golgi staining is a common method for fine-structure labeling of neurons in rodents,non-human primates,and human brains.Although several tissue clearing methods have been used for the clearing of Golgi-stained tissues,the existing methods have long processing times and have not been subjected to detailed immunostaining tests.In view of the above problems,this study aims to establish an ultrafast tissue clearing technology suitable for 3D analysis of large-scale brain structures.The main results are as follows:For rapid clearing of thicker tissue sections for 3D imaging,by testing the tissue clearing effect of various chemical reagents and their combinations,we developed a rapid tissue clearing method based on alkaline solution(AKS,containing 2,2’-thiodiethanol(TDE),DMSO,D-sorbitol,and Tris).The reagent adopts a non-toxic formula and tissue clearing can be accomplished with one-step immersion.Tissue sections with 300-μm-thickness and 1-mm-thickness can be cleared within 5 minutes and 1 hour,respectively.In contrast,conventional clearing methods usually require multiple operation steps and several hours to several days for accomplishing tissue clearing.At the same time,the signals of various fluorescent proteins treated by AKS are significantly stronger than those of tissues treated by various other rapid clearing methods.AKS can be used for long-term high-resolution imaging of weak fluorescent protein-labeled samples.In addition,AKS is compatible with a variety of fluorescent dyes used for immunofluorescence staining,neurocircuit tracing,tissue structure and morphology labeling,thus can be used for 3D imaging of a variety of fluorescent dye-labeled samples.Using AKS,we performed long-time high-resolution imaging of weak fluorescent protein-labelled tissues,long-distance fiber tracking,larger-scale 3D imaging and cell counting of the entire brain area,neural circuit tracing,3D neuromorphic reconstruction,and 3D histopathology imaging.After hundreds of years of validation,the Golgi staining method is still the most classic method to describe neuronal structures,especially dendrites and axons,and dendritic spines.To make Golgi-Cox staining suitable for tissue clearing and 3D morphological analysis,by optimizing Golgi-Cox staining,tissue clearing and 3D immunostaining conditions,an expansion-based method for rapid clearing of Golgi-Cox-stained tissues was developed.This method can rapidly clear 1-mm-thickness Golgi-Cox-stained tissue within 6 h.Cleared Golgi-Cox-stained tissue allows for 3D imaging and is compatible with immunostaining.Using this method,we realized for the first time 3D immunostaining and multiple rounds of immunostaining of Golgi-Cox-stained tissues.By combining the Golgi-Cox staining with tissue clearing and immunostaining,Golgi-Cox-stained tissue could be used for large-volume 3D imaging,identification of cell types of Golgi-Cox-stained cells,and reconstruction of the neural circuits at dendritic spines level.At the same time,these applications were also validated in human brain samples.Conclusion:The alkaline solution-based tissue clearing technique can be used to rapidly acquire 3D data of cell morphology,and the modified expansion-based method can clear the Golgi-Cox-stained tissues,which provides a new methodological support to reveal the fine 3D structure of the nervous system.