Laser fixed-point timing and quantitatively induce venous thrombosis in zebrafish as a general model system to address the essential initial inflammation cytokines

In recent years, a critical clinical problem that cannot be ignored is the incidence of venous thrombosis gradually increasing. Although many animal models of thrombosis have been established, the complex mechanism of thrombosis has not been elucidated. We successfully established a new animal model of venous thrombosis by infrared-pulse laser selectively targeting damage to the venous endothelium of zebrafish tail, resulting in the aggregation of red blood cells and platelets at the site of injury, forming a thrombus, like the formation of thrombi caused by damaged human venous endothelium. o-Dianisidine staining showed increased hemoglobin density at the injury site and decreased hemoglobin density at the heart site. Utilizing laser microdissection technology, we targeted the acquisition of localized thrombus cell clusters for high-throughput transcriptome sequencing. The data were further analyzed through gene set variation analysis (GSVA) and gene set enrichment analysis (GSEA), with the transcriptome data being examined against backgrounds of GO, KEGG, Reactome, and WP databases. Combining these analyses with molecular biology techniques such as RT-qPCR, WISH, and Western Blot, we observed that, compared to the normal group, macrophages were activated, and erythrocyte differentiation was more vigorous post-thrombus formation. Signaling pathways related to cell adhesion and leukocyte migration were activated, and the expression of inflammatory cytokines increased. Notably, IL-6 and TNF-a significantly increased at the thrombus site, while other cytokines such as IL-1ß, IL-10, P-selectin, STAT3, phosphorylated STAT3, p65, and phosphorylated p65 were major players in the inflammatory response during venous thrombosis. The venous thrombosis model established in this study allows a high degree of visualization of thrombosis and provides a feasible and powerful protocol for studying the mechanism of thrombosis. This study may serve as a new venous thrombosis model for exploring the detailed kinetics and underlying mechanisms of thrombosis formation. Overall design: Endothelial cells were laser-damaged at the posterior two somites of the zebrafish anal pore to establish a thrombus model, with the experimental procedure identical to the previous method. Sampling was conducted at the initial stage, peak formation, stabilization, and regression of thrombus at 1h, 3h, 6h, and 12h post-laser injury. The zebrafish larvae subjected to laser damage were sectioned using a sharp blade at five somites below the anal pore, removing the head and tail. The thrombus-forming tissue was sliced into 20 sections with a thickness of 20um each, and these frozen sections were adhered to slides coated with polyethylene naphthalate film (Figure 3A-C). Subsequently, thrombus tissue cells were captured via laser-capture microdissection on frozen sections, and the obtained thrombus tissue cells underwent microRNA extraction. Total RNA was extracted using Invitrogen TRizol reagent (California, USA). Generation of the Illumina RNA sequence library, along with paired end sequencing (300±50 bp) was conducted on an Illumina Hiseq 4000 (LC science, USA) using aliquots (5 mg) of qualified total RNA. Hisat software was used to align compare the sequencing data with the zebrafish reference genome (http://ftp.ensembl.org/pub/release-106/fasta/danio_rerio/dna/Danio_rerio.GRCz11.dna.toplevel.fa.gz). To assess the expression level of all mRNAs, sample reads were initially processed through StringTie, and then the FPKM (fragments per kilobase of transcript per million mapped reads) was calculated using stringtie and edgeR software. Subsequently, differentially expressed genes (DEGs) were identified using the R software package, based on the following criteria: log2 (fold-change) > 1 or <-1 and at p < 0.05. These DEGs were further analyzed for their functions and signaling pathways using Gene Ontology (GO; http://geneontology.org/) and Kyoto Encyclopedia of Genes and Genomes (KEGG; https://www.genome.jp/kegg/).