Longitudinal Changes to Tight Junction Expression and Endothelial Cell Integrity in a Mouse Model of Sterile Corneal Inflammation

Abstract

Citation: Downie LE, Choi J, Lim JKH, Chinnery HR. Longitudinal changes to tight junction expression and endothelial cell integrity in a mouse model of sterile corneal inflammation. Invest Ophthalmol Vis Sci. 2016;57:3477-3484. DOI:10.1167/iovs.15-19005 PURPOSE. We previously reported that applying toll-like receptor (TLR) ligands to an injured cornea induces corneal edema at 24 hours, which subsides by 1 week. We tested the hypotheses that endothelial expression of the tight-junction protein, zonula occludens-1 (ZO-1), would be altered during experimental sterile corneal inflammation and that endothelial cell density (ECD) would remain unaffected.METHODS. Anesthetized C57BL/6J mice received central 1-mm corneal abrasions followed by topical application of saline or cytosine-phosphate-guanosine oligodeoxynucleotide (CpG-ODN, TLR-9 agonist). At 24 hours, 1 week and 4 weeks post treatment, spectral-domain optical coherence tomography images were captured. Eyes were enucleated and processed for zonula occludens-1 (ZO-1) immunofluorescent staining. Corneal flatmounts were analyzed for endothelial ZO-1 expression, cell density, polymegethism, and polymorphism. Corneal stromal inflammatory cell infiltration was evaluated at 4 weeks by immunostaining for CD45.RESULTS. Central corneal thickness (CCT) was increased in CpG-ODN treated eyes at 24 hours, had normalized by 1 week, but was again thickened by 4 weeks. In eyes with CpG-ODN, endothelial cell ZO-1 expression was reduced at 24 hours but returned to normal levels by 1 week. Endothelial cell density was not altered at 24 hours or 1 week. By 4 weeks, only CpG-ODN eyes showed relatively reduced ECD, as well as large numbers of CD45 þ cells in the stroma. Changes to ECD correlated with CCT (r ¼ À0.53, P < 0.01). Compared with naïve controls, more saline-and CpG-ODN-treated eyes exhibited polymegethism.CONCLUSIONS. This study provides novel insights into the interplay between endothelial cell integrity, corneal edema, and chronic stromal leukocyte activation during sterile corneal inflammation in mice.

Anterior segment optical coherence tomography: its application in clinical practice and experimental models of disease

“…In a follow‐up study, the chronic pathological changes induced by topical application of the TLR9 ligand, CpG‐DNA, to the injured mouse cornea was clinically assessed using SD‐OCT. In these studies, elevated corneal thickness was associated with reduced endothelial cell density and morphology, and the presence of giant macrophages in the corneal stroma, four weeks post‐treatment …”

“…In a mouse model of sterile inflammation caused by topical application of endotoxin or CpG‐DNA, corneal oedema, endothelial inflammatory cells (keratic precipitates), anterior chamber cells, and stromal opacities were longitudinally monitored . In these studies, the differences in strain responses were striking, with the corneas of C57BL/6 J mice appearing to have fully recovered by seven days post‐injury, whereas BALB/c mice exhibited signs of ongoing inflammation (corneal opacity and oedema) at the same time point …”

“…Similar to its clinical use, novel applications of AS‐OCT to longitudinally monitor and quantitate the extent of inflammation have been documented in multiple experimental models of ocular inflammation, including uveitis, microbial keratitis, sterile inflammation and dry eye disease . Additionally, AS‐OCT has been used to monitor the resolution of inflammation in experimental models of corneal transplantation and corneal wound healing .…”

“…Unpublished data from the laboratory of the authors of this review indicate that there is a strong correlation between endothelial keratic precipitate counts in histological sections and OCT images. In this study, inflammatory cells were induced into the cornea and anterior chamber using a well‐established model of sterile inflammation, whereby synthetic CpG‐DNA (a mimic of bacterial/viral DNA) was topically applied to the injured mouse cornea . One week post‐treatment, the presence of inflammatory cells adhering to the corneal endothelium can be clearly resolved by in vivo SD‐OCT.…”

Distribution of Corneal TRPV1 and Its Association With Immune Cells During Homeostasis and Injury

“…To induce the nerve injury, a 2 mm trephine was used to mark the central cornea and the epithelium within this zone was debrided using a corneal rust ring remover (Algerbrush II; Alger Equipment Co., Lago Vista, TX, USA). 39 , 40 To prevent dryness of the ocular surface, a 2 µL drop of sterile saline was applied. After one week, the mice were euthanized by lethal injection of Lethabarb (100 mg/kg, intraperitoneal; Lyppard, Keysborough, VIC, Australia).…”