Three-dimensional Microscopic Analysis Of The Vasculature In Whole Mouse Brain And The Distribution Of Tumor-associated Neutrophils In A Glioma Model

The glial vascular unit composed of nerve cells and blood vessels provides sufficient blood supply for active brain regions.In order to further understand how nerve cells and blood vessels interact,it is necessary to quantitatively analyze the relationship between nerve cells and blood vessels.Glioma is one of the most common primary malignant intracranial tumors caused by malignant transformation of intracranial glioblastoma,of which glioblastoma(grade IV)is the most malignant.In the process of tumor development,it is not only related to its own tumor cells,but also to the tumor vasculature and the tumor microenvironment.Glioma,as one of the tumor types with the most abundant blood supply,has abnormally high neovascularization.The 3D visualization of tumor vasculature and quantitative description of vascular characteristics will deepen the understanding of glioma vasculature.Neutrophils are an important component of tumor microenvironment,and also one of the most abundant white blood cells in the circulatory system,accounting for 50%-70%of myeloid white blood cells.The neutrophil group in the tumor microenvironment will differentiate into different subgroups,and some of them will infiltrate into the tumor tissue,which is called tumor-related neutrophils(TANs).In the traditional concept,neutrophils participate in immune regulation and resist virus invasion,which is considered to be the link of inhibiting tumor development.However,more and more evidences show that the inflammatory response of neutrophils plays a positive role in the development and metastasis of tumors.In this paper,the spatial relationship between neuronal cells and blood vessels was analyzed by 3D visualization of whole brain neuronal cells and blood vessels in mice through tissue transparency and light sheet 3D microscopic imaging techniques.And a glioma mouse model was constructed to study the distribution characteristics of tumor blood vessels and TANs.The main studies are as follows:Most existing tissue transparency protocols cause alterations in tissue morphology to varying degrees,and endogenous tissue autofluorescence such as heme and lipofuscin severely interferes with the observation of markers in 3D fluorescence imaging.In this study,tissue transparency reagents were optimized to treat biological samples without causing morphological alteration of the samples.Meanwhile,a decolorization mixture reagent was developed to eliminate the autofluorescence of tissues.The statistical analysis of the volume fraction,mean radius and number of bifurcation points of the blood vessels in the cortical,hippocampal and thalamic regions of the mouse whole brain was completed.The intact brain tissue was obtained by cardiac perfusion of 4%paraformaldehyde,and rapid imaging of the whole mouse brain was achieved by multi-dimensional simultaneous fly-by-scan imaging(VISo R)at 1×1×3.5μm~3 voxel resolution using a tissue transparency method.The two-dimensional image sequences were reconstructed into three-dimensional whole-brain images using the image reconstruction method established in our laboratory to visualize the whole-brain vasculature and nerve cells in mice.A custom Python code was used to segment and extract blood vessels from selected regions of the cortex,hippocampus and thalamus,and analyze their morphological features.The results showed that the parameters of blood vessels were different in different brain regions of mice,and the volume fraction and bifurcation point density of blood vessels were the lowest in the hippocampal region.Simultaneous imaging of blood vessels and nerve cells in the whole brain of mice was achieved.The distance and spatial location between nerve cells and blood vessels were also quantified,and the results showed that the relationship between nerve cells and blood vessels was closest in the mouse brain cortical region.A glioma mouse model was constructed by injecting GL261 glioma cells in C57BL/6mice with fluorescent protein-labeled vascular endothelial cells.The volume fraction,mean radius and total length of blood vessels per unit volume in the tumor region were analyzed comparatively.The tumor areas were divided into central,parenchymal and marginal areas according to the morphological characteristics of the vessels in the tumors,and the distribution characteristics of TANs were statistically analyzed in different areas of the tumors.The results showed that the volume fraction of blood vessels and the total length of blood vessels per unit volume were lower in the central area of the tumor.In contrast,the distribution of TANs was more dense in the margins of the tumor,suggesting that the pro-tumor effect of TANs may be superior to the anti-tumor effect during the development of tumor.In this study,the application of tissue transparency method combined with 3D microscopic imaging technique can achieve rapid imaging of whole brain vasculature and neuronal cells in mice.The quantitative analysis of blood vessels and TANs in the tumor region of the mouse model revealed that glioma blood vessels and neutrophils have a regional distribution.