A Mouse Model for Ad37 Corneal Infection

Abstract

Abstract: : Purpose:Adenoviruses are major human pathogens and a common cause of ocular surface infection. Subgroup D adenoviruses, including Ad8, Ad19, and Ad37, are responsible for significant ocular morbidity as the etiologic agents of epidemic keratoconjunctivitis. We have previously shown in vitro that the earliest host responses to adenovirus infection of keratocytes are controlled by intracellular signaling cascades that activate within minutes of exposure to the virus and result in the expression of chemokines. However, in vivo confirmation of intracellular signaling as the pathogenic mechanism is lacking. A mouse model would offer obvious advantages over in vitro tissue culture models, but human adenoviruses do not replicate in murine cells. We sought to create a mouse model of adenovirus–induced inflammatory gene expression that would not require adenoviral replication. Methods: Intracorneal injection of cesium chloride gradient–purified Ad37 was performed in 6–8 week old female Balb/c mice using sterile glass needles and a CO₂ powered injection system. Corneas were harvested 4 hrs post–injection, and homogenized in TRIzol for RNA isolation. Total tissue RNA was subjected to quantitative real–time PCR for analysis of mRNA expression for select inflammatory mediators. Untouched, needle only, and dialysis buffer–injected corneas were used as controls. Results: Histology of injected corneas demonstrated intrastromal corneal blebs without perforation of Descemets membrane. Real–time PCR analysis of the proinflammatory mediators KC, IP–10, and IL–6 demonstrated on average a 3–5 fold higher expression of mRNA at 4 hrs post–injection in the Ad37–injected corneas compared to buffer–injected corneas. Conclusions: Our approach successfully delivered Ad37 to the corneal stroma of BALB/c mice, and induced reproducible increases in gene expression for several important mediators of inflammation. The injection technique used should allow analysis of the role of host cell signaling even in the absence of adenoviral replication.

Keywords: adenovirus • cornea: stroma and keratocytes • cytokines/chemokines