Generation Of High-Precision And High-Content Whole Mouse Brain Atlases And Its Application In Alzheimer’s Disease Mice

It is of great significance to obtain high-resolution mapping of multiple structures in the whole brain for further understanding of the brain function and the pathogenesis of neurological disorders.The existing imaging techniques and methods are facing great challenges in achieving large-scale and high-resolution synchronous imaging of multiple brain structures Alzheimer’s disease(AD)is one of the neurodegenerative diseases that seriously threaten human life and health.The development of AD is often associated with extensive histological and pathophysiological changes.On the one hand,the importance of cerebrovascular structural and functional abnormalities in the development of AD is increasingly recognized.But at the mesoscopic level,the morphological and anatomical changes of cerebral blood vessels,especially capillaries in AD pathology,remain to be elucidated.On the other hand,a variety of imaging methods have been available to reveal the structural and distributing characteristics of Aβ plaques,and multi-channel fluorescent labeling have also been adopted to observe the effect of Aβ plaque on the basic cerebral structures.However,there is still a lack of high-precision and cross-scale studies on the three-dimensional reconstruction of Aβ plaques and their surrounding environment throughout the brain.In view of these above problems,we developed a whole brain imaging strategy,and employed this strategy to achieve a high-precision atlas of multiple brain structures in AD mice.In the first part,the platform establishing for whole mouse brain imaging technology and subsequent image processing analysis are carried out to achieve the establishment of the whole brain high-precision mapping method.In order to achieve the high-resolution imaging of the whole brain and interpretation of massive complex data following imaging,we firstly accomplished the application of Micro-Optical Sectioning Tomography(MOST)technology and the establishment and improvement of data analysis workflow,which included sample preparation,data acquisition,image preprocessing and optimization,three dimensional(3D)visualization reconstruction and quantitative analysis,and virtual endoscopy.Numerous difficulties had been overcome with respect to enhancement of low SNR image,optimization of image noise,efficient rendering and quantitative analysis of 3D data with high spatial complexity.The visualization of whole brain vascular network in C57BL/6 mice showed that the cortex,thalamus,and hippocampus had their own distinct vascular pattern,and the mean vascular diameter,vascular length density,and vascular volume fraction of the hippocampus were the lowest.The high-precision reconstruction of the entire hippocampus revealed its rake-like vascular distribution pattern.The main blood vessels in the molecular layer of the dentate gyrus(DG-ml)undergo abrupt changes in both diameter and branch angle,presenting a unique comb-like capillary distribution pattern.The virtual endoscopy of individual hippocampal vascular branch allowed to observe the morphology,smoothness,and bifurcation pattern of the inner surface of the vascular lumen from a unique perspective,and could provide intraluminal structural information that are unavailable by conventional visualization methods.In the second part,the high-resolution reconstruction and quantitative analysis of the whole-brain vascular system in APP/PS1 transgenic mice were performed,and the detailed changes of hippocampal vascular distribution pattern and morphological features associated with AD pathology were systematically described at sub-micron resolution for the first time Using the established MOST technology platform,we first obtained the Nissl-stained whole brain datasets of APP/PS1 transgenic AD mice and wild-type mice,and constructed a cross-scale 3D vascular atlas ranging from large vessels to down to smallest capillaries at submicron resolution in the whole mouse brain.Systematic quantitative analysis of the cerebrovascular network of the wild-type and APP/PS1 mice found that the mean vascular diameter and volume fraction of hippocampal vessels in the AD model mice were significantly reduced.Further comparative analysis of different hippocampal subregions revealed that the mean vascular diameter,length density,and volume fraction of the DG-ml showed most significant reduction.The quantitative analysis of the branching pattern of single hippocampal vessel showed that the branch angle in APP/PS1 mice significantly decreased,resulting in the reduced perfusion area of single hippocampal vessel.The virtual endoscopy further revealed that wild-type and APP/PS1 mice had significant differences in roughness and smoothness of the inner vascular lumen.These results demonstrated the capability of high-resolution cross-scale assessment of cerebrovasculature,and systematically revealed the damage of hippocampal microcirculation in AD pathologyIn the third part,the high-precision 3D reconstruction was performed on the whole-brain Nissl staining datasets in 5 × FAD transgenic AD mice,and the whole-brain distribution of Aβplaques based on Nissl staining as well as the high-precision panorama of Aβ plaques and their surrounding soamta,nerve processes,nerve tracts and blood vessels was realized for the first time.Aiming at the problems in signal extraction and image processing caused by complex background and gray heterogeneity of high-throughput bright-field images,a multi-structure signal extraction and synchronous visualization method including "virtual channel splitting"and " feature fusion" is designed,and the high-precision cross-scale whole brain reconstruction of multiple structures is performed.The areas with the highest density of Aβ plaques and the areas with the highest density of large-size plaques were near the entorhinal cortex and the adjacent ventral hippocampal subiculum regions,which implied that Aβ related pathology might begin in these densest regions.In the brain-wide scale,the plaque-dense areas exhibited more somata but less nerve tracts,and localized in the distal end of vessels.In local regions,the visualization and quantitative analysis of cortical areas showed that the plaque-dense areas were close to the deep regions with abundant somata,and the Aβ plaques in the hippocampus were found distributed in the vicinity of cell layers.By viewing at subcellular resolution,the bending or abrupt ending of the nerve processes in the stratum radiatum,as well as distortion or fracture of micro-vessels in the subiculum were visualized.In addition,the sunitinib,sildenafil and cotinine,probably affecting blood vessels,were selected for drug administration experiments and behavioral tests,and then the cotinine-treated mice with the most obvious behavioral changes were selected for multi-structure synchronous visualization.It was found that the A(3 plaques in the cortex were prone to decrease after administration,and there was no obvious changes in the hippocampus.The larger cortical vessels perpendicular to the brain surface were prone to increase after administration,and the volume fraction of cortical vessels increased significantly.However,there was no significant change of the vascular morphological parameters in the hippocampus after administration.These results revealed that the 3D visualization methods based on the MOST technique could be employed to study drug effectsOur study provided a novel method for information extraction and visual reconstruction of high-throughput grayscale images,and achieved high-precision synchronous visualization of multiple structures in the whole brain.The clear presentation of various structural information in the whole brain provides new approaches for in-depth understanding of the anatomical features of the brain under the pathological state of AD.In current study,the clear description of the hippocampal angioarchitecture,the systematic evaluation of the cerebral vasculature of AD mice,and the simultaneous visualization of Aβ plaques and its surrounding multiple structures have not only promoted the in-depth understanding of the basic structure of the mouse brain under normal and AD pathological conditions at mesoscopic level,but also might provide a new reference for the preclinical development of AD-related drugs.