Establishment Of 3D Mesoscopic Imaging Method For Visualizing Fine Blood Vessels And Immune Cells In Liver Lobe

The complex vascular structure and various immune cells in the liver are important for maintaining liver homeostasis.However,it remains a bottleneck to quantify the liver vascular structure and immunocyte distribution at different scales,from liver immunocytes at the micron level to liver vessels at the millimeter level.Optical transparency is an emerging technology for obtaining three-dimensional(3D)structural information about various organs.However,the dense nature of liver tissue with abundant lipids and pigmentation prevents efficient clearing of the liver.Here,a liver-suitable optical clearing method,named liver-CUBIC,was developed for systematic and comprehensive analysis of the liver vascular structure and the dendritic cell(DC)/macrophage distribution and function in intact hepatic lobes.The main conclusions include the following aspects:(1)The liver-CUBIC approach applied lipase-saturated perfusion solution to accelerate the step of liver defatting,which reduced the fluorescence signal loss caused by the longterm action of the transparency reagent.Meanwhile,the improvement of the temperature condition(4 ℃)and p H(9.5)during refractive index matching protected the fluorescence signals from denaturation,especially for the GFP fluorescence signal.Thus,the developed liver-CUBIC approach laid the technical foundation for large-volume imaging of liver vessels and immune cells at single-cell resolution.(2)Based on the liver-CUBIC method,this thesis visualized the intact vasculature structure with different sizes at micrometer resolution and characterized the lobular shape and location in the intact hepatic lobe.More than 72% of the hepatic lobule in the mouse liver tended to be oblate ellipsoids,and the densely distributed sinusoids in the hepatic lobules adopted a scattered honeycomb feature.The average sinusoidal length,radius and tortuosity was 24.91 μm,2.92 μm,1.13,respectively.In the carbon tetrachloride(CCl4)-induced fibrotic liver,the hepatic lobule tended to adopt a portal lobular feature.Fibrosis induced a 1.4% increase in sinusoidal density,a 4.8% decrease in sinusoidal radius,a 7.2%decrease in sinusoidal tortuosity,and a more than 55% increase in 4 ~ 7 edge loop in the sinusoidal network.Quantifying sinusoidal morphology and topology in the hepatic lobules offered new approaches to investigating vascular function in liver physiology and pathology.(3)Based on the liver-CUBIC method,this research investigated the distribution feature of immunocytes in liver physiology and pathology.The distribution of CD11c+ cells followed a lognormal pattern in the classical lobule in the healthy liver,in which 40% of the CD11c+ cells were distributed at the portal vein and 2% of CD11c+ cells were distributed surrounding the central vein.However,in the CCl4-induced chronic liver injury,60% of the CD11c+ cells accumulated at the central vein in the portal lobule.In the murine nonalcoholic steatohepatitis model,the number of CD11c+ cells was increased by 125%,and the average distance from CD11c+ cells to a central vein in the lobule was decreased from221 μm to 127 μm.The uncovering of CD11c+ cells’ distribution in intact hepatic lobules provided intuitive evidence for immunological mechanisms in liver diseases.(4)Based on the liver-CUBIC approach,this research investigated tumor-immune cellvascular crosstalk in the intact lobe in murine colorectal liver metastasis.It demonstrated that the accumulation of CX3CR1+CCR2+F4/80+ macrophages at metastatic foci was closely related to the metastasis’ s location and volume in the liver.Furthermore,interruption of CCL2/CCR2 signaling blocked CX3CR1+CCR2+F4/80+ macrophage recruitment and inhibited the early colonization and growth of liver metastases.These results suggested that metastatic cells might shape an immunosuppressive environment via mobilizing CX3CR1+CCR2+F4/80+ macrophage recruitment around the early colorectal metastases.In summary,a fast and fluorescence-preserving liver-clearing approach was developed for the 3D mesoscopic visualization of liver anatomic structure and immunity.This approach provided a visual and intuitive platform for revealing the liver’s fine vasculature structure and immunocyte arrangement.Exploring and quantifying the correlation between early colorectal micrometastasis and CX3CR1+CCR2+ macrophage in intact liver lobe provided new knowledge for tumor immunity in colorectal liver metastasis.