Establishment Of Early Diabetes Retinopathy Model In Tree Shrews And Preliminary Study On Its Pathogenesis

Objectives: Diabetic retinopathy(DR)is one of the main causes of blindness in adults.It is a multifactorial disease whose mechanism is unknown and there is no effective treatment for early lesions.Animal models that effectively replicate DR characteristics are needed to investigate the underlying molecular mechanisms.At present,a variety of animal models of DR have been developed,including rodents,dogs,cats,pigs,rabbits,zebrafish and non-human primates,but all have their limitations,and cannot fully reconstruct the complete pathophysiology of neuronal and vascular changes at various stages of DR.Therefore,there is an urgent need to establish animal models with similar symptoms to human DR.Tree shrews are more similar to humans in terms of eyeball structure and metabolism than rodents,dogs,rabbits,cats and other experimental animals.This study was to use the high-fat and high-sugar feed joint chain urea with streptozotocin(STZ)established diabetic mellitus(DM)tree shrews,studying whether the retina of DM tree shrews has the same morphological and molecular changes as that of early human DR,to explore whether tree shrew can be used as a new animal model of DR.The m RNA in the retina of DM tree shrews was detected to screen the target genes and related signaling pathways that may be involved in the occurrence of DR,and the possible target genes were verified in vivo and in vitro to provide basic data for studying the pathogenesis of DR.Methods:(1)DM tree shrews’ model were constructed by high-fat and high-sugar diet combined with STZ(100 mg/kg),which was the experimental group;normal tree shrews were the control group.Fasting blood-glucose(FBG),body weight,urine glucose,fasting insulin(FINS)and lipoprotein were observed after modeling.(2)Morphological changes in the retina of DM tree shrews were observed by fundus color photography,retinal capillary pavement and transmission electron microscopy,and pathological changes in the retina,liver,kidney,pancreas and spleen were observed by HE staining.(3)The expression levels of vascular endothelial growth factor(VEGF)and apoptosis protein Bax in DM tree shrews were detected by Western Blot.(4)Highthroughput transcriptome sequencing was used to detect m RNA expression profiles in the retina of DM tree shrews at the 20 th week,screen for significantly differentially expressed genes and key signal pathways,and some genes with more obvious differences(Mybpc2,PSAT1 and ST6GALNAC2)were selected for RT-q PCR verification.(5)Primary retinal ganglion cells(RGCs)were isolated and cultured using enzymatic digestion to establish an in vitro model of high glucose injury in RGCs,and the expression level of PSAT1 in RGCs after high glucose intervention was verified in vitro.Results: After modeling with high fat and high sugar diet combined with STZ,FBG in the experimental group increased significantly after the second week compared with the control group(P < 0.01);After 14 weeks,the body weight decreased compared with the control group(P < 0.05);Urine sugar was qualitatively positive;At the 8th week,FINS in the experimental group was significantly higher than that in the control group(P < 0.05);At 20 weeks,TC,HDL-C,and LDL-C significantly increased in the experimental group(P < 0.05),while TG did not change significantly.The fundus color photography results showed that the animal’s lens became turbid at the third month.Through retinal HE staining,it was found that at the 20 th week after modeling,the thickness of retinal nerve fiber layer(RNFL)and ganglion cell layer(GCL)in the experimental group became thinner than that in the control group(P < 0.05);the retinal capillary pavement showed ghost pericytes and acellular capillaries in the retina.The results of transmission electron microscopy showed that the DM tree shrew retinal vessels were damaged,and the pericytes and endothelial cells were moderately edematous,and the tightly connected dense areas were shortened;VEGF(P < 0.01)and Bax protein were significantly upregulated(P < 0.05)in the experimental group by Western Blot.By transcriptome sequencing,we identified 178 significantly different genes,including 93 up-regulated genes and 85 down-regulated genes.Mybpc2,PSAT1 and ST6GALNAC2 genes were selected for RT-q PCR experiments to compare the trends of these genes in DM tree shrews and normal tree shrews,and the trends were the same as the sequencing results.KEGG was mainly enriched in MAPK signaling pathway,PPAR signaling pathway and insulin resistance,etc.The primary RGCs were successfully isolated and cultured,and the PSTA1 expression level of RGCs after high glucose intervention was found to be consistent with the animal level.Conclusion: 1.High-fat and high-sugar diets combined with STZ injections successfully established DM tree shrews,which mainly showed signs of polyphagia,polyphagia,polyuria and weight loss,and blood tests showed significant hyperglycemia,hyperlipidemia and insulin resistance,consistent with diabetes mellitus.2.At the 20 th week,the retina of DM tree shrews showed features of neurodegeneration and vascular damage in early DR.Retinal RNFL and GCL layers become thinner,retinal ghost pericytes and acellular capillaries appear,retinal ultrastructure and blood vessels are damaged,and VEGF and Bax are upregulated.Tree shrews can be used as animal models for studying early DR.3.During the formation of DR,multiple m RNAs are differentially expressed in the DM tree shrew retina,and multiple signaling pathways enriched by these m RNAs and their target genes.These m RNAs and their target genes are enriched in several signaling pathways,which may be involved in the development of early DR pathological process,and the specific regulatory mechanism in DR still needs to be further investigated.4.We successfully isolated and cultured primary RGCs of tree shrew,and verified in vitro that the expression level of PSAT1 in RGCs after high glucose intervention is consistent with the animal level,indicating that PSAT1 is associated with damage to RGCs and early DR,suggesting that PSAT1 may be involved in regulating the development of early stages of DR.