The Establishment Of Zebrafish Three-dimensional Mass Spectrometry Imaging Method And Its Application In The Study Of NPC1 Disease

Mass spectrometry imaging is a new type of visual analysis and detection technology.It has been recognized as an effective tool for detecting and identifying a variety of complex molecules in biological samples,and is used in life sciences,biomedicine,environmental toxicology and other fields widely.In recent years,there have been more and more diseases caused by abnormal lipid metabolism in the body,and people have paid more and more attention to the role of lipid metabolism in the body.There have been many reports using two-dimensional mass spectrometry imaging technology to study the spatial distribution of lipids in biological tissue samples.Through two-dimensional imaging analysis of lipids in a single tissue section sample,it is possible to understand the changes in lipids in the course of disease development,so that people can have more understanding of the lipid function and lipid regulation process in different organs.However,most organisms in nature are three-dimensional,so it is difficult to characterize the lipid changes of the entire body with a single cross-section.Therefore,the establishment of a three-dimensional mass spectrometry imaging method for whole animals is of great significance for studying the impact of lipid-related diseases on the body.In this study,we have established a three-dimensional matrix assisted laser desorption ionization（MALDI）mass spectrometry imaging method for lipid analysis of whole zebrafish.By acquiring MALDI mass spectrometry imaging data of serial sections of samples,then use professional processing software to superimpose the data to construct a three-dimensional zebrafish image.This method can provide information on the spatial distribution of target molecules in multiple organs,and can observe the distribution of multiple molecules in different organs at the same time.It can reflect the spatial distribution of target compounds more truly and completely.In this experiment,the method was applied to the study of NPC1gene mutation(npc1^-/-)lipid content and spatial distribution changes in zebrafish,and it was found that npc1^-/-zebrafish have a variety of sphingolipids and phospholipids showing significant differences and these differential lipids have organ specificity.For example,compared with wild-type(npc1^+/+)zebrafish,the content of lysophosphatidylethanolamine（LPE）in the spinal cord of npc1^-/-zebrafish is reduced,while the content of sphingomyelin（SM）is increased.Reduced PE content in the intestine,while the content of lysophosphatidylinositol（LPI）,phosphatidylglycerol（PG）and phosphatidylinositol（PI）in the liver and spleen increased.The zebrafish three-dimensional MALDI mass spectrometry imaging method is used to the study of NPC1 disease,we have obtained the overall situation of lipid changes in different organs and functional systems of npc1^-/-zebrafish.It can provide further understanding of the relationship between abnormal lipid accumulation in NPC1disease and subsequent clinical symptoms（such as neurological degeneration and hepatosplenomegaly）,and provide new ideas for the study of lipid-related diseases.