Zebrafish caudal fin amputation models the effects of diabetes on inflammation and regeneration
Aim: In this study, we aim to establish a diabetic zebrafish model and examine the effects of high glucose on inflammation and tissue regeneration. Materials and Methods:  Tübingen strain zebrafish (wild type, WT), Tg (mpo: EGFP), and Tg (coro1a: EGFP) zebrafish were employed. Caudal fin amputation was performed to study tissue regeneration and inflammation. Results:  Our results showed that high glucose increased the recruitment of neutrophils and macrophages after amputation. Most importantly, limb regeneration was severely impaired after amputation in zebrafish larvae exposed to 222 mM glucose for 14 days. In addition, inflammatory factors IL-1ß, IL-6, TNF-a, VCAM-1, and MCP-1, which are closely related to the development of diabetic wound, increased significantly in caudal fin amputation after glucose exposure. The p38 inhibitor SB203580 can reduce the expression of inflammatory factors and promote regeneration in high glucose. It showed that caudal fin amputation after glucose exposure resulted in genes enrichment of several pathways involved in amino acid metabolism, lipid metabolism, and inflammation based on high-throughput transcriptome sequencing. Conclusions:  Overall, this experimental system using zebrafish larvae provides a wide spectrum of genetic and molecular profile recapitulating human diabetes which will be beneficial for basic research of inflammation and regeneration in diabetic background and promote translational research for diabetes therapy. Overall design: Zebrafish embryos were placed in 30?ml of culture medium in petri plates with 130 mM or 222 nM D-glucose at 6 hours post fertilization (hpf) and control group were maintained in regular culture medium. All embryos were cultured in incubator at 28.5 ?. The culture medium was replaced at half volume every day.  Caudal fin amputation was performed after glucose exposure for 14 days. Briefly, the larvae were fixed with 3% carboxymethyl cellulose in the culture dish after anesthesia with 0.03% tricaine. The caudal fin was carefully removed at the distal end of the spinal cord of the zebrafish larvae with a 1 mm sapphire blade, without spinal cord injury. After caudal fin amputation, the zebrafish larvae were placed back to original culture condition. Total RNA was extracted using Invitrogen TRizol reagent (California, USA), and generation of the Illumina RNA sequence library and paired end sequencing (300±50 bp) were performed on an Illumina NovaSeq 6000 using aliquots (5 mg) of qualified total RNA. Hisat software was used to compare the sequencing data with the zebrafish referenced genome. To evaluate the expression level of all mRNAs, we first realized sample reads through StringTie, and then calculated the FPKM (fragments per kilobase of transcript per million mapped reads) using stringtie and edgeR software. Finally, the differentially expressed genes (DEGs) were screened by the R software package according to the following criteria: log2 (fold-change) > 1 or <-1 and at p < 0.05. These DEGs were used for further analysis of functions and signaling pathways using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes.