Evaluation Of Corneal/conjunctival Injury And Detection Of Pyroptosis In The Model Of Alkali Burn Of Ocular Surface

Objective 1.To establish a mouse ocular surface alkali burn model simulating human ocular surface alkali burn;2.;To evaluate the corneal/conjunctival injury and observe the changes of neovascularization and fibrosis of ocular surface tissue after alkali burn;3.To explore whether pyroptosis is involved in the injury response of ocular surface alkali burn.Methods Part I:10 ul of 1 N NaOH were applied to the right eye of 6-week-old C57BL/6J mice under general anesthesia to produce a total ocular surface alkali burn.Corneal opacity score,ocular surface staining score,corneal neovascularization length measurement were performed on the 3rd,5th,7th,14th and 22nd days.Part II:Establish a mouse ocular surface alkali burn model,observe and collect corneal tissue and conjunctival tissue on the 3rd,5th,7th,14th and 22nd day respectively.The mRNA levels of VEGF、MMP-2、MMP-9 and α-SMA in cornea and conjunctiva were detected by Real-time quantitative PCR,and western blot methods detected the expression levels of VEGF、MMP-2、MMP-9 and α-SMA protein in corneal tissue and conjunctival tissue.Mice were then sacrifificed and histology with hematoxylin-eosin on eyeball was used to observe the inflammatory infiltration of mouse ocular surface on days 7th,14th and 22nd day.Part III:Establish a mouse ocular surface alkali burn model,observe and collect corneal tissue and conjunctival tissue on the 3rd,5th,7th,14th and 22nd day respectively.The mRNA levels of NLRP3、caspase-1、GSDMD、IL-1β in cornea and conjunctiva were detected by Real-time quantitative PCR,and western blot methods detected the expression levels of NLRP3、caspase-1、GSDMD、IL-1β protein in corneal tissue and conjunctival tissue.Resluts 1.The degree of corneal opacity in mice was the most serious on the 14th day after alkali burn,and then decreased.The epithelial defect area of ocular surface tissue reached the highest value in 5-7 days,and then the epithelium healed gradually.The corneal neovascularization of mice gradually invaded the cornea over time,and the corneal neovascularization invaded the center of the cornea on the 14th day after injury.2.After alkali burn of mouse ocular surface,the results of real-time fluorescence quantitative PCR and WB showed the mRNA and protein expression levels of neovascularization related indexes VEGF,MMP-2,MMP-9 and fibrosis related indexes α-SMA increased significantly.In conjunctival tissue,angiogenesis related indexes VEGF,MMP-2 and MMP-9 began to rise on the 3rd-5th day and continued to the 22nd day,α-SMA began to rise on day 3 and continued to day 22.In corneal tissue,angiogenesis related indexes VEGF,MMP-2 and MMP-9 began to rise on the 3rd-5th day and continued to the 22nd day,α-SMA began to rise on day 5-7 and continued to day 22.3.After alkali burn of mouse ocular surface tissue,the fluorescence quantitative PCR results and WB results of ocular surface tissue showed the expression levels of mRNA and protein of the relevant indexes of pyroptosis NLRP3,caspase-1,gsdmd and IL-1 β were significantly higher than those in the blank control group.Conclusions 1.1 0ul 1 N NaOH applying on mouse ocular surface for 30s can establish a relatively stable and reliable mouse ocular surface alkali burn model simulating human ocular surface alkali burn.2.It is preliminarily confirmed that pyroptosis may be involved in ocular surface alkali burn.